Noel M. Delos Santos

Characterization of the Residues in Helix 8 of the Human β1-Adrenergic Receptor That Are Involved in Coupling the Receptor to G Proteins

Several key amino acids within amphipathic helix 8 of the human β1-adrenergic receptor (β1-AR) were mutagenized to characterize their role in signaling by G protein-coupled receptors. Mutagenesis of phenylalanine at position 383 in the hydrophobic interface to histidine (F383H) prevented the biosynthesis of the receptor, indicating that the orientation of helix 8 is important for receptor biosynthesis. Mutagenesis of aspartic acid at position 382 in the hydrophilic interface to leucine (D382L) reduced the binding and uncoupled the receptor from G protein activation. Mutagenesis of the basic arginine residue at position 384 to glutamine (R384Q) or to glutamic acid (R384E) increased basal and agonist-stimulated adenylyl cyclase activities. R384Q and R384E displayed features associated with constitutively active receptors because inverse agonists markedly reduced their elevated basal adenylyl cyclase activities. Isoproterenol increased the phosphorylation and promoted the desensitization of the Gly389 or Arg389 allelic variants of the wild type β1-AR but failed to produce these effects in R384Q and R384E, because these receptors were maximally phosphorylated and desensitized under basal conditions. In contrast to the membranous distribution of the wild type β1-AR, R384Q and R384E were localized mostly within intracellular punctate structures. Inverse agonists restored the membranous distribution of R384Q and R384E, indicating that they recycled normally when their constitutive internalization was blocked by inverse agonists. These data combined with computer modeling of the putative three-dimensional organization of helix 8 indicated that the amphipathic character of helix 8 and side chain projections of Asp382 and Arg384 within the hydrophilic interface might serve as a tethering site for the G protein.

Renal function in survivors of nonsyndromic Wilms tumor treated with unilateral radical nephrectomy: Renal Function in UWT After Nephrectomy

Partial nephrectomy is considered by some for children with unilateral Wilms tumor (UWT) to avoid the theoretical complication of renal insufficiency. In the current study, the authors evaluated the prevalence of hypertension and impaired renal function in long‐term survivors of nonsyndromic UWT who were treated without nephrotoxic chemotherapy or ionizing radiation.

Renal survival in pediatric patients with IgA nephropathy.

Sirs, We commend the efforts of Ronkainen et al. [1] in obtaining long-term follow-up data on IgA nephropathy in children, particularly since renal survival data for pediatric patients remain limited. We agree with the authors that the difficulties are considerable with respect to locating former pediatric patients who may have transitioned to providers of adult care or who have simply stopped seeking medical care. The authors determined predicted kidney survival for 55 Finnish patients diagnosed with IgA nephropathy prior to age 18 years. Predicted renal survival rates in this cohort from time of onset of symptoms were 93 and 87% at 10 and 20 years, respectively. In 1995, our group described renal survival in a cohort of 103 pediatric patients of Caucasian (88), African-American (12), Asian (2), and Native American (1) race diagnosed in Memphis, Tennessee, and Lexington, Kentucky. [2] The predicted kidney survival rates from the time of biopsy were 85% at 10 years and 73% at 20 years. Since Berger and Hinglais did not describe IgA nephropathy until 1968 [3], and the condition was not routinely diagnosed in U.S. centers prior to 1972, actual 20-year follow-up was possible for only 5 of 103 patients. We would like to take this opportunity to update renal survival data from 67 Caucasian patients from Tennessee to indicate how they compare to the Finnish data. In this group, 69% were male. The mean age at time of biopsy was 10.8±3.3 years (range 2.1–17.4 years) and mean follow-up from time of biopsy was 9.3±8.2 years (range 0.003– 25.47). Six patients (9%) progressed to end-stage renal disease. Tenand 20-year renal survival rates predicted using the Kaplan-Meier method were 91 and 80%, respectively (Fig. 1). Nine (13%) of our patients had more than 20 years of follow-up. Ronkainen et al. reported survival from time of onset, while we report survival from time of biopsy. They also did not include patients with less than 10 years of follow-up, while we included such patients. Nevertheless, the predicted kidney survival rates for the Finnish cohort and Caucasian patients in the Memphis cohort were quite similar. Pediatr Nephrol (2007) 22:317–318 DOI 10.1007/s00467-006-0303-3

Mutagenesis within Helix 6 of the Human β1-Adrenergic Receptor Identifies Lysine324 as a Residue Involved in Imparting the high-affinity Binding State of Agonists

Competition binding isotherms for agonists to G protein-coupled receptors (GPCR) display high and low binding affinities. Mutagenesis of lysine at position 324 in helix 6 of the wild-type (WT) human β1-adrenergic receptor (β1-AR) generated mutant receptors that had GTP-insensitive single low-affinity binding sites for agonists and reduced potencies of full or partial agonists in stimulating adenylyl cyclase. Unlike the WT β1-AR, intrinsic activities of full and partial agonists in activating the Lys324→ Ala β1-AR (K324A) mutant were correlated with their binding affinities to the K324A mutant. In assays, such as agonist-mediated phosphorylation and recycling, the K324A mutant and the WT β1-AR behaved similarly. However, in fluorescence resonance energy transfer assays that determined the proximity between the WT β1-AR or the K324A mutant to Gsα, there were significant differences. The conceptual framework of the ternary complex model could not adequately account for the behavior of the K324A mutant except under assumptions of low receptor-G protein binding affinities. The single low-affinity binding site of the K324A mutant to isoproterenol was converted by the C-terminal 11-amino-acid peptide of Gsα, which acts a GDP-bound Gsα mimic, to high- and low-affinity sites. Based upon the three-dimensional architecture of the human β1-AR, the distance between Lys324 and the Asp/Glu-Arg-Tyr motif in helix 3 was the shortest among the various amino acids in helix 6. These findings indicate that Lys324 lies in a groove between helices 3 and 6, and its mutagenesis generates a mutant receptor with very low binding affinity for the GDP-bound isoform of Gs.

Comprehensive renal function evaluation in patients treated for synchronous bilateral Wilms tumor

OBJECTIVES The purpose of this study was to perform a comprehensive assessment of long-term renal function in patients treated at our institution for synchronous bilateral Wilms tumor (BWT) and to determine the optimal method for estimating glomerular filtration rate (eGFR). METHODS Surgical approach, adjuvant therapy, and pathology reports were reviewed for patients with at least six months follow-up from definitive surgery. eGFRs, as assessed by the Schwartz and Chronic Kidney Disease in Children (CKiD) formulas, were compared to measured GFR (mGFR) determined by 99mTc-DTPA scanning. Urine studies, including microalbumin, β-microglobulin, and FENa were also reviewed. RESULTS Forty-two patients were identified. Of 36 living patients, 28 (77.8%) had greater than 6months follow-up, with a median overall follow-up of 5.2years (range: 1.4-13.4). The median mGFR was 97mL/min/1.73m2, while the median eGFRSchwartz and eGFRCKiD were 103.3mL/min/1.73m2 and 79.7mL/min/1.73m2, respectively, (p=0.13 and p=0.75, compared to mGFR). Eleven (39.3%) patients had at least one abnormal urine study (microalbumin >30μg/g creatinine, n=3; β-2 microglobulin >133μg/g creatinine, n=9; FENa>1%, n=4). CONCLUSIONS In our series, few patients had an abnormally low GFR. Neither method for estimating GFR gave a significantly different result from measured GFR, suggesting that the Schwartz equation is adequate, although specific urine tests may be more sensitive for detecting subtle renal dysfunction. LEVEL OF EVIDENCE Level IV - retrospective case series with no comparison group.

Post-infectious acute glomerulonephritis with predominant mesangial deposition of IgA

Postinfectious acute glomerulonephritis (PIAGN), including poststreptococcal acute glomerulonephritis (PSAGN), is commonly seen in children and usually poses no diagnostic difficulties. Horita et al. reported an interesting case of histologically confirmed superimposition of PSAGN during IgA nephropathy (IgAN) in the December issue of your journal. We are reporting the case of a child with Henoch– Schönlein purpura (HSP) nephritis with a history and histology suggestive of concurrent PIAGN. The patient was an 8-year-old Caucasian boy with fever, abdominal pain, diarrhea, and gross hematuria for 3 days. He had had a red rash on his legs about 3 weeks earlier and an intermittent sore throat for 6 months. The family history was unremarkable. Examination showed an afebrile child with a blood pressure of 148/96mmHg, but otherwise unremarkable. Laboratory values included white cell count 11.8 ¥ 10/mm, hemoglobin 12.6g/dl, and platelet count 307 ¥ 10/mm. Blood urea nitrogen (BUN) was 29mg/dl, creatinine 0.7mg/dl, and albumin 2.9g/dl. His complement 3 level was 36 mg/dl. Antistreptolysin-O and anti-DNAse-B titres were both negative. Urinalysis showed 2+ protein and 3+ blood with 30–40 red cells per high-power field (rbc/hpf) and dysmorphic morphology. The patient was discharged with an antibiotic and an antihypertensive, but was admitted again 5 days later with a petechial and purpuric rash on his legs and scrotal swelling. A renal biopsy showed increased tuft cellularity in all glomeruli due to intrinsic cell

Pediatric C1q nephropathy and incidental proteinuria.

Sirs, We had previously reported our experiences of the clinical features and outcomes of C1q nephropathy in 20 children in Memphis, Tennessee [1]. Dr. Nishida et al. reported three patients with C1q nephropathy and asymptomatic proteinuria that were identified by the school urinary screening program in Japan in the December 2005 issue of Pediatric Nephrology [2]. In their study, 94 of the 193 renal biopsies were performed for asymptomatic proteinuria. They identified three patients with asymptomatic proteinuria with biopsies that fulfilled the diagnostic criteria of C1q nephropathy. While all of these patients had mild proteinuria without obvious clinical symptoms, they all had less than ten red blood cells per high power field in the urine sediments under the microscope. The renal biopsies from all patients showed membranoproliferative histology. While 40% of our patients presented with nephrotic syndrome, three (15%) African–American patients (one female and two males) presented with incidental proteinuria. They were diagnosed with abnormal urinalysis during sport-related physical examinations. They were all normotensive and none of them had any other symptoms such as hematuria or swelling at presentation that would have suggested any renal disease. They had either no blood or trace amounts of blood in the urine at presentation. However, in contrast to the patients from the study by Dr. Nishida, all of our patients had nephrotic range proteinuria and the average urine protein to creatinine ratio at presentation was 4.12. The average serum creatinine was 1.1 mg/dL and the average estimated glomerular filtration rate (GFR) by the Schwartz formula was 87.1 ml/ min/1.73 m [3]. The renal biopsies all showed focal segmental glomerulosclerosis. Two patients were subsequently treated solely with ACE inhibitor, while the other patient was also treated with oral steroids and mycophenolate mofetil. At last follow-up, all three patients still had persistent proteinuria but were negative for blood in their urine. Two of them had serum creatinine levels of 1.5 mg/ dL and the other had a level of 0.9 mg/dL. One of the patients had an estimated GFR of less than 50 ml/ min/1.73 m at last follow-up. In addition to more severe degrees of proteinuria at presentation, the histologies of the renal biopsies of our patients were very different from the cases reported by Dr. Nishida, and our patients with incidental proteinuria had less favorable outcomes than the group from Japan. The difference in ethnicity may account for this discrepancy. Since we do not routinely screen the urine of our children in the United States, the actual incidence of asymptomatic proteinuria and of C1q nephropathy is unknown. Although our data support Dr. Nishima’s observation that a considerable number of patients with asymptomatic proteinuria may have C1q nephropathy, unlike their patients, not all of ours had benign clinical outcomes. Larger studies of well-characterized patients and longer follow-up times are necessary for a better understanding of this clinical entity.

Elucidation of Renal Scars in Children With Vesicoureteral Reflux Using Contrast-Enhanced Ultrasound: A Pilot Study

Introduction Vesicoureteral reflux is a common disorder in children but can result in kidney scarring following acute pyelonephritis. The gold standard diagnostic to detect renal scars in children is 99mTc-dimercaptosuccinic acid (DMSA) scintigraphy. DMSA has a number of limitations including radiation exposure, need for sedation, and radiotracer supply shortages. Contrast-enhanced ultrasound (CEUS) is a technique whereby biocompatible microspheres of inert gas are administered i.v. that reflect ultrasonography sound waves and do not involve radiation. Because the contrast agent is rapidly cleared, contrast images must be obtained within minutes of administration. CEUS has been used in a variety of organ systems, but its use in pediatric kidney diseases is limited. Methods In this study, we performed CEUS in 7 children with documented renal scars by radiographic imaging consistent with reflux nephropathy. Results In all subjects, CEUS detected all previously known radiologic abnormalities as well as detecting new areas of hypoenhancing renal parenchyma. None of the patients experienced any serious adverse events. Discussion This study represents the first report of using CEUS to characterize renal scars in children with reflux nephropathy. We conclude that CEUS is a highly sensitive, rapid, and cost-effective diagnostic imaging modality for detecting and monitoring renal scars in children with vesicoureteral reflux.

Fever and Rash

revealed a left pleural effusion and left lower lobe atelectasis/consolidation. Fluid obtained from ultrasound-guided thoracentesis had 930 white blood cells/mm with 39% neutrophils, 3% bands, 38% lymphocytes, lactate dehydrogenase 196 IU/L, total protein 2.5 g/dL, pH 8.0, and cultures negative for bacteria. Because of persistent symptoms despite parental doxycycline, ceftriaxone (1 g daily) was added. After 10 days of doxycycline and 7 days of ceftriaxone, his skin rash resolved but he remained febrile (38-40°C) and continued to have generalized fatigue and bilateral lower extremity edema. Serum titers for tick-borne illnesses (Table 1) drawn after 2 weeks of illness were positive for Ehrlichia chaffeensis (immunoglobulin G of 1:512 and immunoglobulin M of 1:80). As no other titers for infectious entities were positive, rifampin was added to treat a possible resistant infection. When fever and proteinuria (4+ proteinuria) persisted despite treatment, the possibility of an underlying systemic illness was entertained. A 24-hour urine specimen was collected that contained 7700 mg (194.2 mg/m/h) protein (Table 1). A renal biopsy was performed, which confirmed the diagnosis of membranous lupus nephritis (International Society of Nephrology/Renal Pathology Society class V) (Figure 3). Other laboratory parameters that supported the diagnosis of systemic lupus erythematosus (SLE) included a positive antinuclear antibody (ANA) titer of 4.79 with a homogenous pattern together with a positive antibody to double-stranded DNA and low serum complement levels (C3 and C4). Taken together, his clinical symptoms and laboratory parameters confirmed the diagnosis of SLE. He was started on prednisone 40 mg twice daily with immediate resolution of his fatigue and fever and a more gradual resolution of the proteinuria and peripheral edema. Case Report

Tularemia infection in a pediatric patient with chronic renal insufficiency and inguinal lymphadenopathy

Abstract: Tularemia is a rare zoonosis caused by the gram-negative coccobacillus Francisella tularensis. We describe a pediatric patient with chronic renal insufficiency who presented with high fever and regional lymphadenopathy caused by tularemia. This case highlights the fact that F. tularensis infection must be considered when investigating a fever of unknown origin, especially in the presence of a history of animal exposure.