Titilayo O. Ilori

Acute calcineurin inhibition with tacrolimus increases phosphorylated UT-A1

UT-A1, the urea transporter present in the apical membrane of the inner medullary collecting duct, is crucial to the kidneys ability to concentrate urine. Phosphorylation of UT-A1 on serines 486 and 499 is important for plasma membrane trafficking. The effect of calcineurin on dephosphorylation of UT-A1 was investigated. Inner medullary collecting ducts from Sprague-Dawley rats were metabolically labeled and treated with tacrolimus to inhibit calcineurin or calyculin to inhibit protein phosphatases 1 and 2A. UT-A1 was immunoprecipitated, electrophoresed, blotted, and total UT-A1 phosphorylation was assessed by autoradiography. Total UT-A1 was determined by Western blotting. A phospho-specific antibody to pser486-UT-A1 was used to determine whether serine 486 can be hyperphosphorylated by inhibiting phosphatases. Inhibition of calcineurin showed an increase in phosphorylation per unit protein at serine 486. In contrast, inhibition of phosphatases 1 and 2A resulted in an increase in UT-A1 phosphorylation but no increase in pser486-UT-A1. In vitro perfusion of inner medullary collecting ducts showed tacrolimus-stimulated urea permeability consistent with stimulated urea transport. The location of phosphorylated UT-A1 in rats treated acutely and chronically with tacrolimus was determined using immunohistochemistry. Inner medullary collecting ducts of the acutely treated rats showed increased apical membrane association of phosphorylated UT-A1 while chronic treatment reduced membrane association of phosphorylated UT-A1. We conclude that UT-A1 may be dephosphorylated by multiple phosphatases and that the PKA-phosphorylated serine 486 is dephosphorylated by calcineurin. This is the first documentation of the role of phosphatases and the specific site of phosphorylation of UT-A1, in response to tacrolimus.

RNA-seq analysis of glycosylation related gene expression in STZ-induced diabetic rat kidney inner medulla.

The UT-A1 urea transporter is crucial to the kidneys ability to generate concentrated urine. Native UT-A1 from kidney inner medulla (IM) is a heavily glycosylated protein with two glycosylation forms of 97 and 117 kDa. In diabetes, UT-A1 protein abundance, particularly the 117 kD isoform, is significantly increased corresponding to an increased urea permeability in perfused IM collecting ducts, which plays an important role in preventing the osmotic diuresis caused by glucosuria. However, how the glycan carbohydrate structure change and the glycan related enzymes regulate kidney urea transport activity, particularly under diabetic condition, is largely unknown. In this study, using sugar-specific binding lectins, we found that the carbohydrate structure of UT-A1 is changed with increased amounts of sialic acid, fucose, and increased glycan branching under diabetic conditions. These changes were accompanied by altered UT-A1 association with the galectin proteins, β-galactoside glycan binding proteins. To explore the molecular basis of the alterations of glycan structures, the highly sensitive next generation sequencing (NGS) technology, Illumina RNA-seq, was employed to analyze genes involved in the process of UT-A1 glycosylation using streptozotocin (STZ)—induced diabetic rat kidney. Differential gene expression analysis combining with quantitative PCR revealed that expression of a number of important glycosylation related genes were changed under diabetic conditions. These genes include the glycosyltransferase genes Mgat4a, the sialylation enzymes St3gal1 and St3gal4 and glycan binding protein galectin-3, -5, -8, and -9. In contrast, although highly expressed in kidney IM, the glycosyltransferase genes Mgat1, Mgat2, and fucosyltransferase Fut8, did not show any changes. Conclusions: In diabetes, not only is UT-A1 protein abundance increased but the proteins glycan structure is also significantly changed. UT-A1 protein becomes highly sialylated, fucosylated and branched. Consistently, a number of crucial glycosylation related genes are changed under diabetic conditions. The alteration of these genes may contribute to changes in the UT-A1 glycan structure and therefore modulate kidney urea transport activity and alleviate osmotic diuresis caused by glucosuria in diabetes.

Urine concentration in the diabetic mouse requires both urea and water transporters

The regulation of the inner medullary collecting duct (IMCD) urea transporters (UT-A1, UT-A3) and aquaporin-2 (AQP2) and their interactions in diabetic animals is unknown. We investigated whether the urine concentrating defect in diabetic animals was a function of AQP2, the UT-As, or both transporters. UT-A1/UT-A3 knockout (UT-A1/A3 KO) mice produce dilute urine. We gave wild-type (WT) and UT-A1/A3 KO mice vasopressin via minipump for 7 days. In WT mice, vasopressin increased urine osmolality from 3,000 to 4,550 mosmol/kgH(2)O. In contrast, urine osmolality was low (800 mosmol/kgH(2)O) in the UT-A1/A3 KOs and remained low following vasopressin. Surprisingly, AQP2 protein abundance increased in UT-A1/A3 KO (114%) and WT (92%) mice. To define the role of UT-A1 and UT-A3 in the diabetic responses, WT and UT-A1/A3 KO mice were injected with streptozotocin (STZ). UT-A1/A3 KO mice showed only 40% survival at 7 days post-STZ injection compared with 70% in WT. AQP2 did not increase in the diabetic UT-A1/A3 KO mice compared with a 133% increase in WT diabetic mice. Biotinylation studies in rat IMCDs showed that membrane accumulation of UT-A1 increased by 68% in response to vasopressin in control rats but was unchanged by vasopressin in diabetic rat IMCDs. We conclude that, even with increased AQP2, UT-A1/UT-A3 is essential to optimal urine concentration. Furthermore, UT-A1 may be maximally membrane associated in diabetic rat inner medulla, making additional stimulation by vasopressin ineffective.

Phosphatase inhibition increases AQP2 accumulation in the rat IMCD apical plasma membrane

Vasopressin triggers the phosphorylation and apical plasma membrane accumulation of aquaporin 2 (AQP2), and it plays an essential role in urine concentration. Vasopressin, acting through protein kinase A, phosphorylates AQP2. However, the phosphorylation state of AQP2 could also be affected by the action of protein phosphatases (PPs). Rat inner medullas (IM) were incubated with calyculin (PP1 and PP2A inhibitor, 50 nM) or tacrolimus (PP2B inhibitor, 100 nM). Calyculin did not affect total AQP2 protein abundance (by Western blot) but did significantly increase the abundances of pS256-AQP2 and pS264-AQP2. It did not change pS261-AQP2 or pS269-AQP2. Calyculin significantly enhanced the membrane accumulation (by biotinylation) of total AQP2, pS256-AQP2, and pS264-AQP2. Likewise, immunohistochemistry showed an increase in the apical plasma membrane association of pS256-AQP2 and pS264-AQP2 in calyculin-treated rat IM. Tacrolimus also did not change total AQP2 abundance but significantly increased the abundances of pS261-AQP2 and pS264-AQP2. In contrast to calyculin, tacrolimus did not change the amount of total AQP2 in the plasma membrane (by biotinylation and immunohistochemistry). Tacrolimus did increase the expression of pS264-AQP2 in the apical plasma membrane (by immunohistochemistry). In conclusion, PP1/PP2A regulates the phosphorylation and apical plasma membrane accumulation of AQP2 differently than PP2B. Serine-264 of AQP2 is a phosphorylation site that is regulated by both PP1/PP2A and PP2B. This dual regulatory pathway may suggest a previously unappreciated role for multiple phosphatases in the regulation of urine concentration.

Oxidative Balance Score and Chronic Kidney Disease

Background: The oxidative balance score (OBS) is a composite estimate of the overall pro- and antioxidant exposure status in an individual. The aim of this study was to determine the association between OBS and renal disease. Methods: Using the Reasons for Geographic and Racial Differences in Stroke cohort study, OBS was calculated by combining 13 a priori-defined pro- and antioxidant factors by using baseline dietary and lifestyle assessment. OBS was divided into quartiles (Q1-Q4) with the lowest quartile, Q1 (predominance of pro-oxidants), as the reference. Multivariable logistic regression and Cox proportional hazards models were used to estimate adjusted ORs for albuminuria defined as urine albumin/creatinine ratio (ACR) >30 mg/g, macroalbuminuria defined as ACR >300 mg/g and chronic kidney disease (CKD) defined as estimated glomerular filtration rate <60 ml/min/1.73 m2 according to the Chronic Kidney Disease Epidemiology Collaboration and hazards ratios for end-stage renal disease (ESRD), respectively. Results: Of the 19,461 participants analyzed, 12.9% had albuminuria and 10.1% had CKD at baseline; over a median follow-up of 3.5 years (range 2.14-4.32 years), 0.46% developed ESRD. Higher OBS quartiles were associated with lower prevalence of CKD (OR vs. Q1: Q2 = 0.93 [95% CI 0.80-1.08]; Q3 = 0.90 [95% CI 0.77-1.04] and Q4 = 0.79 [95% CI 0.67-0.92], p for trend <0.01). The associations between OBS and albuminuria (p for trend 0.31) and incident ESRD (p for trend 0.56) were not significant in the fully adjusted models. Conclusions: These findings suggest that higher OBS is associated with lower prevalence of CKD. Lack of association with ESRD incidence in the multivariable analyses indicates that temporal relation between OBS and renal damage remains unclear.

The Arteriovenous Fistula: An Often Overlooked Precipitant of HighOutput Heart Failure

Heart failure usually occurs in the setting of a low cardiac output, however in rare cases it may be associated with a high cardiac output. We present a rare and intriguing case of a patient with end-stage renal disease (ESRD) on hemodialysis that developed high output heart failure from his arteriovenous fistula (AVF). A 36 year old African American male with ESRD secondary to hypertension, on hemodialysis for six years, presented to emergency room with a history of chest pain and shortness of breath. He was diagnosed with congestive heart failure secondary to coronary artery disease. His transthoracic echo showed an ejection fraction of 65% and both the stress test and cardiac enzymes were negative for ischemia. Further workup revealed a cardiac output of 10.6 L/min and access flow of 2.37 L/min. We made a diagnosis of high output heart failure secondary to the AVF. He was taken to surgery for a minimally invasive limited ligation endoluminal-assisted revision (MILLER) procedure after which his symptoms resolved. High output heart failure, though a rare complication of AVF placement is an important differential in hemodialysis patients who present with symptoms of congestive heart failure. An accurate diagnosis will prevent morbidity and unnecessary hospital admissions.

Oxidative Balance Score and the Risk of End-Stage Renal Disease and Cardiovascular Disease

Background: Oxidative balance score (OBS) is a composite measure of oxidative stress-related exposures. The aim of this study was to investigate the association between OBS, end-stage renal disease (ESRD), and cardiovascular disease (CVD). Methods: Using data from the Chronic Renal Insufficiency Cohort, we calculated the main exposure OBS by summing up 12 apriori-defined pro- and antioxidant factors obtained from the diet history questionnaire and lifestyle assessment. We divided OBS into quartiles (Q1-Q4), with Q1 (predominance of pro-oxidants) as the reference. We analyzed OBS quartiles as an ordinal variable. Crude and adjusted hazards ratios (HRs) and 95% CIs were estimated using Cox proportional hazards models for time to ESRD and CVD. Results: Compared to Q1, Q4 (high antioxidant) was associated with ESRD in the crude model (HR 1.35, 95% CI 1.08-1.69) and adjusting for age, sex, and race (HR 1.36, 95% CI 1.09-1.71) but not in the fully adjusted model (HR 1.12, 95% CI 0.84-1.51). HR of ESRD increased as the OBS quartiles increased in the crude model (ptrend < 0.05) but not in the fully adjusted model (ptrend = 0.30). Compared to Q1, Q4 was associated with CVD in the crude (HR 1.33, 95% CI 1.06-1.68) but not adjusted models. The HR of CVD increased with an increase in OBS quartiles in the crude model (ptrend < 0.05). Conclusion: The reverse association between OBS and progression to ESRD suggests that perhaps the effect of oxidative balance-related exposure is different in the setting of established chronic kidney disease.

Comparison of Outcomes between Individuals with Pure and Mixed Lupus Nephritis: A Retrospective Study.

Introduction Lupus nephritis (LN) is a serious organ manifestation of systemic lupus erythematosus. Histologic overlap is relatively common in the six pathologic classes (I to VI) of LN. For example, mixed proliferative LN (MPLN) often includes features of classes III & V or classes IV & V combined. We performed a comparative evaluation of renal outcomes in patients with MPLN to patients with pure proliferative LN (PPLN) against pre-specified renal outcomes, and we also identified predictor of clinical outcomes among those with PPLN and MPLN. Hypothesis Individuals with MPLN will have worse short-term renal outcomes compared to those with PPLN. Methods We retrospectively reviewed 278 adult LN patients (≥18 years old) identified from an Emory University Hospital registry of native renal biopsies performed between January 2000 and December 2011. The final analytic sample consisted of individuals with a diagnosis of PPLN (n = 60) and MPLN (n = 96). We analyzed differences in clinical and laboratory characteristics at baseline. We also assessed associations between LN category and renal outcomes (complete remission and time to ESRD) with logistic and Cox proportional hazards models within two years of baseline. Results The study population was predominantly female (83.97%) and African American (71.8%) with a mean age of 33.4 years at baseline. Over a median follow up of 1.02 years, we did not find any statistically significant associations between MPLN and the development of ESRD or remission when compared to patients with PPLN (adjusted HR = 0.30, 95% CI = 0.07, 1.26). Conclusion There was no association between mixed or pure histopathologic features of LN at presentation and rate of complete or partial remission but higher baseline eGFR was associated with a lower probability of complete remission among patients with lupus nephritis.

Racial and Ethnic Disparities in Graft and Recipient Survival in Elderly Kidney Transplant Recipients

To investigate racial and ethnic differences in graft and recipient survival in elderly kidney transplant recipients.

Dietary Oxidative Balance Scores and Biomarkers of Inflammation among Individuals with and without Chronic Kidney Disease

Background: Oxidative stress and inflammation are proposed mechanisms of nonspecific kidney injury and progressive kidney failure. Higher dietary oxidative balance scores (OBS) are associated with lower prevalence of chronic kidney disease (CKD). Methods: We investigated the association between OBS and biomarkers of inflammation using data from the Reasons for Geographic and Racial Differences in Stroke (REGARDS) study. Nutrient estimates from the Block Food Frequency Questionnaires were used to define tertiles of 11 pro- and antioxidant factors. Points for each OBS component were summed, with a higher score indicating predominance of antioxidant exposures. Multivariable linear regression models were used to estimate the association between OBS and biomarkers of inflammation (interleukin-6 [IL-6], interleukin-8 [IL-8], interleukin-10 [IL-10], fibrinogen, C-reactive protein [CRP], white blood cell count, and cystatin C). An interaction term was included to determine if associations between OBS and inflammatory markers differed between individuals with and without CKD. Results: Of 682 participants, 22.4% had CKD. In adjusted models, OBS was associated with CRP and IL-6. For every 5-unit increase in OBS, the CRP concentration was –15.3% lower (95% CI: –25.6, –3.6). The association of OBS with IL-6 differed by CKD status; for every 5-unit increase in OBS, IL-6 was –10.7% lower (95% CI: –16.3, –4.7) among those without CKD, but there was no association among those with CKD (p = 0.03). Conclusion: This study suggests that a higher OBS is associated with more favorable levels of IL-6 and CRP, and that the association of OBS and IL-6 may be modified by CKD status.