Comparative Analysis of Protein Quantification by the SomaScan Assay versus Orthogonal Methods in Urine from People with Diabetic Kidney Disease.

To our knowledge, calibration curves or other validations for thousands of SomaScan aptamers are not publicly available. Moreover, the abundance of urine proteins obtained from these assays is not routinely validated with orthogonal methods (OMs). We report an in-depth comparison of SomaScan readout for 23 proteins in urine samples from patients with diabetic kidney disease (n = 118) vs OMs, including liquid chromatography-targeted mass spectrometry (LC-MS), ELISA, and nephelometry. Pearson correlation between urine abundance of the 23 proteins from SomaScan 3.2 vs OMs ranged from -0.58 to 0.86, with a median (interquartile ratio, [IQR]) of 0.49 (0.18, 0.53). In multivariable linear regression, the SomaScan readout for 6 of the 23 examined proteins (26%) was most strongly associated with the OM-derived abundance of the same (target) protein. For 3 of 23 (13%), the SomaScan and OM-derived abundance of each protein were significantly associated, but the SomaScan readout was more strongly associated with OM-derived abundance of one or more "off-target" proteins. For the remaining 14 proteins (61%), the SomaScan readouts were not significantly associated with the OM-derived abundance of the targeted proteins. In 6 of the latest group, the SomaScan readout was not associated with urine abundance of any of the 23 quantified proteins. To sum, over half of the SomaScan results could not be confirmed by independent orthogonal methods.

Association of Tubular Solute Clearance with Symptom Burden in Incident Peritoneal Dialysis.

BACKGROUND AND OBJECTIVES: Residual kidney function is important to the health and wellbeing of patients with ESKD. We tested whether the kidney clearances of proximal tubular secretory solutes are associated with burden of uremic and heart failure symptoms among patients on peritoneal dialysis with residual kidney function. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We enrolled 29 patients on incident peritoneal dialysis with residual urine output >250 ml daily. We used targeted liquid chromatography-mass spectrometry to quantify plasma, 24-hour urine, and peritoneal dialysate concentrations of ten tubular secretory solutes. We calculated the kidney and peritoneal dialysis clearances of each secretory solute, creatinine, and urea, and we estimated a composite kidney and peritoneal secretion score. We assessed for uremic symptoms using the Dialysis Symptom Index and heart failure-related symptoms using the Kansas City Cardiomyopathy Questionnaire. We used linear regression to determine associations of composite secretory solute clearances and GFRurea+Cr with Dialysis Symptom Index symptom score and Kansas City Cardiomyopathy Questionnaire summary score. RESULTS: Mean residual kidney clearances of creatinine and urea were 8±5 and 9±6 ml/min per 1.73 m2, respectively, and mean GFRurea+Cr was 8±5 ml/min per 1.73 m2. The residual kidney clearances of most secretory solutes were considerably higher than creatinine and urea clearance, and also, they were higher than their respective peritoneal dialysis clearances. After adjustments for age and sex, each SD higher composite kidney secretion score was associated with an 11-point lower Dialysis Symptom Index score (95% confidence interval, -20 to -1; P=0.03) and a 12-point higher Kansas City Cardiomyopathy Questionnaire score (95% confidence interval, 0.5- to 23-point higher score; P=0.04). Composite peritoneal dialysis secretion score was not associated with either symptom assessment. CONCLUSIONS: Residual kidney clearances of secretory solutes are higher than peritoneal dialysis clearances. Kidney clearances of secretory solutes are associated with patient-reported uremic and heart failure-related symptoms.

Urine RAS components in mice and people with type 1 diabetes and chronic kidney disease.

The pathways implicated in diabetic kidney disease (DKD) are largely derived from animal models. To examine if alterations in renin-angiotensin system (RAS) in humans are concordant with those in rodent models, we measured concentration of angiotensinogen (AOG), cathepsin D (CTSD), angiotensin-converting enzyme (ACE), and ACE2 and enzymatic activities of ACE, ACE2, and aminopeptidase-A in FVB mice 13-20 wk after treatment with streptozotocin (n = 9) or vehicle (n = 15) and people with long-standing type 1 diabetes, with (n = 37) or without (n = 81) DKD. In streptozotocin-treated mice, urine AOG and CTSD were 10.4- and 3.0-fold higher than in controls, respectively (P < 0.001). Enzymatic activities of ACE, ACE2, and APA were 6.2-, 3.2-, and 18.8-fold higher, respectively, in diabetic animals (P < 0.001). Angiotensin II was 2.4-fold higher in diabetic animals (P = 0.017). Compared with people without DKD, those with DKD had higher urine AOG (170 vs. 15 µg/g) and CTSD (147 vs. 31 µg/g). In people with DKD, urine ACE concentration was 1.8-fold higher (1.4 vs. 0.8 µg/g in those without DKD), while its enzymatic activity was 0.6-fold lower (1.0 vs. 1.6 × 109 RFU/g in those without DKD). Lower ACE activity, but not ACE protein concentration, was associated with ACE inhibitor (ACEI) treatment. After adjustment for clinical covariates, AOG, CTSD, ACE concentration, and ACE activity remained associated with DKD. In conclusion, in mice with streptozotocin-induced diabetes and in humans with DKD, urine concentrations and enzymatic activities of several RAS components are concordantly increased, consistent with enhanced RAS activity and greater angiotensin II formation. ACEI use was associated with a specific reduction in urine ACE activity, not ACE protein concentration, suggesting that it may be a marker of exposure to this widely-used therapy.

An Optimized Method for Protein Extraction from OCT-Embedded Human Kidney Tissue for Protein Quantification by LC-MS/MS Proteomics.

The existing biobanks of remnant tissue from clinically indicated kidney biopsies are attractive potential reservoirs for quantification of clinically relevant human tissue proteins by quantitative proteomics. However, a significant caveat of this strategy is that the tissues are often preserved in optimal cutting temperature (OCT) medium. Although OCT is an effective method of preserving the morphologic and immunohistological characteristics of tissues for later study, it significantly impacts efforts to quantify protein expression by liquid chromatography-tandem mass spectrometry methods. We report here a simple, reproducible, and cost-effective procedure to extract proteins from OCT-embedded tissue samples. Briefly, the excess frozen OCT medium was scraped before thawing from the tissue specimens stored at -80°C for ∼3 months. The tissue samples were homogenized and diethyl ether/methanol extraction was performed to remove the remaining OCT medium. The recovered protein was denatured, reduced, and alkylated. The second step of protein extraction and desalting was performed by chloroform/methanol/water extraction of denatured proteins. The resultant protein pellet was trypsin-digested and the marker proteins of various kidney cellular compartments were quantified by targeted selective reaction monitoring proteomics. Upon comparison of peptide signals from OCT-embedded tissue and flash-frozen tissue from the same donors, both individual protein quantities, and their interindividual variabilities, were similar. Therefore, the approach reported here can be applied to clinical reservoirs of OCT-preserved kidney tissue to be used for quantitative proteomics studies of clinically relevant proteins expressed in different parts of the kidney (including drug transporters and metabolizing enzymes).

The role of proinsulin and insulin in the diagnosis of insulinoma: a critical evaluation of the Endocrine Society clinical practice guideline.

CONTEXT: An end of fast insulin ≥ 3 µIU/mL and a proinsulin concentration ≥ 5 pmol/L have been suggested as useful cutoffs for the diagnosis of insulinoma. OBJECTIVE: The main objective was to evaluate the diagnostic performance of an end of fast insulin concentration ≥ 3 µIU/mL and an end of fast proinsulin concentration ≥ 5 pmol/L. DESIGN: The design was a case-control series. SETTING: The setting was a tertiary-care center. PATIENTS: Fifty-six subjects with a positive 48-hour supervised fast had an insulinoma between June 2000 and April 2011. During this same time period, a diagnosis of insulinoma was excluded in 29 subjects who underwent a supervised fast. INTERVENTION: 48-hour supervised fast. MAIN OUTCOME MEASURE: The main outcome measures were serum insulin concentration and plasma proinsulin concentration. RESULTS: Ninety-one percent of the patients with an insulinoma had a measured insulin concentration ≥5 µIU/mL at the end of fast. The sensitivity increased to 98% if the threshold to define inadequate insulin suppression was lowered to ≥3 µIU/mL. The median (interquartile range) end of fast proinsulin was 100 (53-270) pmol/L for cases and 6.8 (4.2-12.0) pmol/L for controls. An end of fast proinsulin value of ≥ 5 pmol/L could not distinguish cases from controls (59% false positive rate). All patients with an insulinoma (sensitivity 100%) and none of the control subject (specificity 100%) had end of fast proinsulin concentration ≥ 27 pmol/L. CONCLUSIONS: Using a current insulin assay 9% of insulinoma cases end the supervised fast with an insulin concentration below 5 µIU/mL. Inadequate insulin suppression defined using a threshold of ≥ 3 µIU/mL increases the sensitivity of the test. The value of the proinsulin test lies in its unique ability to distinguish cases from controls. A proinsulin concentration of ≥22 pmol/L best discriminates cases from controls. Reliance on an end of fast proinsulin cutoff value of 5 pmol/L does not augment sensitivity but greatly reduces specificity of the test.

Insulin resistance is a sufficient basis for hyperandrogenism in lipodystrophic women with polycystic ovarian syndrome.

CONTEXT: The lipodystrophies (LD) are characterized by metabolic abnormalities (insulin resistance, hypertriglyceridemia, and diabetes) and a polycystic ovarian syndrome (PCOS) phenotype. Therapeutic administration of leptin improves insulin sensitivity and the metabolic features. OBJECTIVE: The objective of the study was to investigate whether the PCOS features are corrected by increasing insulin sensitivity as a function of leptin treatment. DESIGN: This was a prospective, open-label trial using leptin replacement in various forms of lipodystrophy. SETTING: The study was performed at the Clinical Center at the National Institutes of Health. PATIENTS OR OTHER PARTICIPANTS: Twenty-three female patients with LD were enrolled in a leptin replacement trial from 2000 to the present. Different parameters were assessed at baseline and after 1 yr of therapy. INTERVENTION(S): Patients were treated with leptin for at least 1 yr. MAIN OUTCOME MEASURE(S): We evaluated free testosterone, SHBG, and IGF-I at baseline and after 1 yr of leptin. RESULTS: Testosterone levels decreased from 3.05 ±0.6 ng/ml at baseline to 1.7 ±0.3 ng/ml (P = 0.02). SHBG increased from 14.5 ±2 to 25 ±3.5 nmol/liter after 1 yr of leptin therapy. There were no significant changes in the levels of gonadotropins and ovarian size as a result of leptin replacement therapy. IGF-I increased significantly after leptin therapy from 150 ±14 to 195 ±17. There was a significant decrease in triglycerides and glycosylated hemoglobin in the context of reduced insulin requirements. CONCLUSIONS: In the present study, we show that LD may be a model for the common forms of PCOS and that the endocrine features are corrected by leptin therapy, which reduces insulin resistance.