Podocyte Glutamatergic Signaling Contributes to the Function of the Glomerular Filtration Barrier

Podocytes possess the complete machinery for glutamatergic signaling, raising the possibility that neuron-like signaling contributes to glomerular function. To test this, we studied mice and cells lacking Rab3A, a small GTPase that regulates glutamate exocytosis. In addition, we blocked the glutamate ionotropic N-methyl-d-aspartate receptor (NMDAR) with specific antagonists. In mice, the absence of Rab3A and blockade of NMDAR both associated with an increased urinary albumin/creatinine ratio. In humans, NMDAR blockade, obtained by addition of ketamine to general anesthesia, also had an albuminuric effect. In vitro, Rab3A-null podocytes displayed a dysregulated release of glutamate with higher rates of spontaneous exocytosis, explained by a reduction in Rab3A effectors resulting in freedom of vesicles from the actin cytoskeleton. In addition, NMDAR antagonism led to profound cytoskeletal remodeling and redistribution of nephrin in cultured podocytes; the addition of the agonist NMDA reversed these changes. In summary, these results suggest that glutamatergic signaling driven by podocytes contributes to the integrity of the glomerular filtration barrier and that derangements in this signaling may lead to proteinuric renal diseases.It is widely recognized that most glomerular diseases are characterized by defects of the filtration barrier, where podocytes play a central role. Mutations of single podocyte proteins have been found at the basis of human nephrotic syndromes,¹ and podocyte deletion of the same molecules causes heavy proteinuria in experimental models.^2–8Podocytes are highly ramified cells: From the cell body depart a number of primary processes, further originating secondary foot processes. Starting from these features, it has been demonstrated that podocytes share numerous similarities with neurons: They both are terminally differentiated cells, have a common cytoskeletal organization, and have a common machinery of process formation.⁹ Furthermore, a number of expression-restricted proteins, such as nephrin,² Neph1 and Neph2,¹⁰ GLEPP1,¹¹ CAT3 and EAAT2,¹² synaptopodin,¹³ drebrin,¹⁴ and Sam68-like-MP2,¹⁵ specifically belong to the podocyte and the neuron.Our group has contributed to this line of research, initially by describing in podocytes the presence of Rab3A, a small GTPase that is mostly enriched in synaptic vesicles because it tightly modulates highly regulated exocytosis by acting through a number of effector molecules, including rabphilin-3a and Synapsin-I.¹⁶ After finding that in podocytes, as it occurs in neurons, Rab3A associates to glutamate-containing vesicles along cell processes, we discovered that podocytes are equipped with a complete neuron-like glutamatergic signaling system.¹⁷ We described that podocytes possess functional synaptic-like microvesicles and renal glomeruli express cognate glutamate transporters and receptors. These properties strengthened the analogies between podocytes and neurons and offered a rational interpretation to the biochemical similarity of foot process and synaptic adhesion complexes¹⁷; however, the role played by glutamate signaling in podocytes remained unanswered, and nothing was known about its relevance to podocyte and glomerular homeostasis. To get more details on the requirement of this neuron-like system of signaling by podocytes, we first conducted a preliminary analysis on its temporal appearance during podocyte differentiation. Then we studied conditions in which it was altered, on the vesicle and on the receptor side. The vesicular component was analyzed by studying the consequences of the absence of Rab3A. On the receptor side, we antagonized the ionotropic N-methyl-d-aspartate receptor (NMDAR), that we found present in human and rodent glomeruli, as well as in podocyte cell cultures.¹⁷ Both Rab3A and NMDAR1 glomerular synthesis were also confirmed by in situ hybridization (Supplemental Figure 1) and by microarray expression data.¹⁷     

Glomerular Filtration Rate during Enflurane Anaesthesia

Pre- and preoperative determination of glomerular filtration rate (GFR) was performed in 10 patients operated on during enflurane anaesthesia. The mean GFR decreased significantly by 21%. The decrease in GFR is of the same order of magnitude as that reported for most general anaesthetics.     

Phase Separation of MAGI2-Mediated Complex Underlies Formation of Slit Diaphragm Complex in Glomerular Filtration Barrier

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三种肾小球滤过率检测方法的实用性探讨

目的：以^99mTc—DPTA血浆清除率为标准，对24h内生肌酐清除率（Ccr）、Cookeroft—Gault（CG）方程和简化MDRD（modification of diet in renal disease）方程进行比较，评价三种方程在慢性肾脏疾病（CKD）患者中的应用。方法：选择139例各种慢性肾脏疾病患者，将Ccr、CG方程和简化MDRM方程估算的肾小球滤过率（GFR）用体表面积（BSA）标准化，与BSA标准化的^99mTc—DTPA测得的GFR（^99mTc—GFR）在不同CKD分期进行比较。结果：CKD第一至第五期：Ccr与^99mTc—GFR相关系数r分别为：0．79、0．71、0．64、0．59、0．52；Ccr在ROC曲线下面积平均为0．79。CG—GFR与^99mTc—GFR相关系数r分别为：0．85、0．78、0．72、0．67、0．61；CG—GFR在ROC曲线下面积平均为0．83。MDRD—GFR与^99mTc—GFR相关系数r分别为：0．83、0．76、0．69、0．65、0．59；MDRD—GFR在ROC曲线下面积平均为0．82。在CKD不同分期三种方程的GFR估算值与^99mTc—GFR差异均有显著意义（P均〈0．001）。结论：三种方程的GFR估算值与^99mTc—GFR均有较好的相关性和ROC曲线下面积，以CG方程最好，其次为简化MDRD方程，Ccr最低，但三种方程估算值与^99mTc—GFR测定值差异均有显著意义。上述方程直接应用于我国CKD患者时，应对其进行适当修正。     

Formulas Estimating Glomerular Filtration Rate in the Evaluation of Living Kidney Donor Candidates: Comparison of Different Formulas With Scintigraphy-Measured Glomerular Filtration Rate

IntroductionEstimation of kidney function is crucial in the evaluation of living kidney donor candidates. Despite the multitude of glomerular filtration rate (GFR) formulas, no equation is universal, and none were validated in the population of kidney donors. Novel biomarkers, including beta trace protein (BTP) and cystatin C, are studied to help estimate GFR and improve the safe qualification of living kidney donors.AimThis study compares the accuracy of different formulas that estimate GFR with reference scintigraphy-measured GFR in the population of living kidney donor candidates.Material and MethodsThis study enrolled 30 healthy living kidney donor candidates. GFR was measured using the following 11 different formulas. For reference, GFR was assessed using 99m-Technetium-diethylenetriaminepentaacetic acid.ResultsThe accuracy of estimation was generally low in all formulas. The strongest correlation between measured GFR (mGFR) and estimated GFR (eGFR) was achieved by the Nankivell formula (R = 0.47, P = .009); however, in the group of patients with a body mass index of >25 kg/m², only the equations based on BTP had a statistically significant correlation with mGFR: White (R = 0.59; P = .016) and Poge (R = 0.53; P = .035). Bland-Altman plots revealed wide limits of agreement between eGFRs and mGFR in all groups of patients.ConclusionIn living kidney donor candidates, GFR estimation formulas should be chosen individually. White formula, which is based on BTP, may be a promising tool in estimating GFR in overweight potential living kidney donor candidates. More than 1 formula and personalized choice of GFR estimation method regarding the given patient should be performed in qualification of kidney donors.     

Donor Kidney Glomerular Filtration Rate and Post-Transplant Graft Function

This study aimed to estimate the relationship between the single kidney glomerular filtration rate (SKGFR) of a planned kidney transplant and the subsequent graft function and survival of living related kidney recipients (LKRs). Of 180 LKRs with the graft functioning for more than a year, 70 patients without delayed graft function (DGF) or acute rejection (AR) were selected for the study. According to SKGFR, assessed by 99mTcDTPA, the patients were allocated into Group 1, receiving kidney with SKGFR < 50 mL/min (32 patients), and Group 2, with SKGFR > 50 mL/min (38 patients). The database included donor, recipient and transplant variables. No significant difference was found between the patient and graft survival rate, creatinine clearance (CCr) and the rate of CCr change between the groups. Additional evaluation revealed no significant influence of the ratio of SKGFR and the recipient's body weight/size on patient and graft outcome. The analysis of factors of influence on patient and graft survival and function revealed the major influence of nonimmunological factors but not of SKGFR of the transplanted kidney. Our study did not confirm the influence of SKGFR on graft function and survival in the LKRs without DGF and AR but the limited number of patients must not be disregarded.     

Phosphoproteomic Analysis Reveals Regulatory Mechanisms at the Kidney Filtration Barrier

Diseases of the kidney filtration barrier are a leading cause of ESRD. Most disorders affect the podocytes, polarized cells with a limited capacity for self-renewal that require tightly controlled signaling to maintain their integrity, viability, and function. Here, we provide an atlas of in vivo phosphorylated, glomerulus-expressed proteins, including podocyte-specific gene products, identified in an unbiased tandem mass spectrometry–based approach. We discovered 2449 phosphorylated proteins corresponding to 4079 identified high-confidence phosphorylated residues and performed a systematic bioinformatics analysis of this dataset. We discovered 146 phosphorylation sites on proteins abundantly expressed in podocytes. The prohibitin homology domain of the slit diaphragm protein podocin contained one such site, threonine 234 (T234), located within a phosphorylation motif that is mutated in human genetic forms of proteinuria. The T234 site resides at the interface of podocin dimers. Free energy calculation through molecular dynamic simulations revealed a role for T234 in regulating podocin dimerization. We show that phosphorylation critically regulates formation of high molecular weight complexes and that this may represent a general principle for the assembly of proteins containing prohibitin homology domains.The kidney filter consists of three layers: fenestrated endothelial cells, the glomerular basement membrane, and podocytes.¹ Damage to any of these compartments becomes clinically evident as proteinuria and the development of kidney disease.² Of particular importance for the regulation of podocyte biology through signaling is the slit diaphragm, a specialized intercellular junction that bridges the 40-nm gap in between foot processes of neighboring podocytes. It also serves as a signaling platform regulating podocyte function. Mutations in genes encoding for components of the slit diaphragm, such as nephrin,³ podocin,⁴ CD2AP,⁵ and TRPC6,^6,7 are important causes of genetic forms of proteinuria. Alteration of these proteins results in defective signaling causing podocyte dysfunction, progressive glomerulosclerosis, and kidney failure. The slit diaphragm protein complex is a lipid-multiprotein supercomplex.⁸ Of central importance to the integrity and function of the protein complex is the prohibitin homology (PHB) domain protein podocin,⁹ which forms multimeric complexes and is required to control signal transduction through associated transmembrane proteins.^10,11Signaling processes governing podocyte function, integrity, and survival largely depend on signaling processes involving phosphorylation.^12,13 Comprehensive analyses of the signaling events in podocytes in vivo have been hampered by the fact that interference with these signaling cascades by genetic deletion often results in massively disrupted and dysfunctional podocytes. One of the primary aims of this study was to use phosphoproteomics to analyze thousands of phosphorylation sites in native murine glomeruli within single samples. Within this study, we show that this approach allows the introduction of new concepts into signaling processes at the kidney filtration barrier.     

Assessing Glomerular Filtration Rate by Estimation Equations in Kidney Transplant Recipients

Surveillance of glomerular filtration rate (GFR) is crucial in the management of kidney transplant recipients. With especial emphasis on serum creatinine (SCr) calibration assay, we assessed the performance of estimation equations as compared to iothalamate GFR (iGFR) in 209 patients using the modification of diet in renal disease (MDRD), Nankivell and Cockcroft-Gault methods. Fifty-five percent of patients were treated with a calcineurin inhibitor (CNI) and all were taken trimethroprim-sulfametoxazole at the time of SCr measurement. The mean iGFR was 44 ± 26 mL/min/1.73 m². The MDRD equation showed a median difference of 0.9 mL/min/1.73 m² with 53% of estimated GFR within 20% of iGFR. Median differences were 7.5 and 7.0 mL/min/1.73 m² for Nankivell and Cockcroft-Gault formulas, respectively. The accuracy of the Nankivell and Cockcroft-Gault formulas was such that only 38% and 37% of estimations, respectively, fell within 20% of iGFR. The performance of all equations was not uniform throughout the whole range of GFR, with some deterioration at the extremes of GFR levels. In addition, good performance of the MDRD equation was seen in subjects taking CNI. In conclusion, the overall performance of the MDRD equation was superior to the Nankivell and Cockcroft-Gault formulas in renal transplant recipients including subjects treated with CNI.     

中国肾小球滤过率评估方程在慢性肾小球肾炎患者的适用性评价

目的：评价现有在中国人群基础上开发的肾小球滤过率（GFR）评估方程在慢性肾小球肾炎患者的适用性。方法：选择143例慢性肾小球肾炎患者,用中国方程、瑞金方程、MDRD1方程和简化MDRD方程,分别计算GFR值,与^99mTc—DTPA测的GFR（sGFR）进行比较。结果：Bland—Altman分析显示MDRD1方程和瑞金方程估计的GFR和sGFR的一致性较好,但所有各方程估计的GFR和出FR的一致性限度均超过事先规定的专业界值。线性回归结果显示,MDRD1方程和瑞金方程估测的GFR与X轴的斜率较其他方程更接近0。在所有方程中,MDRD1方程和中国9方程偏差较小,瑞金方程估测GFR30％符合率和50％符合率均最高,但瑞金方程估测GFR30％符合率依然低于70％。在慢性肾脏病不同分期中,瑞金方程和MDRD1方程较其他方程有较小的偏差和更优的准确性。结论：当血肌酐的测定方法为酶法时,如直接应用现有在中国人群基础上开发的肾小球滤过率评估方程评估慢性肾小球肾炎患者肾功能,可能会产生明显的偏差。     

Suitability of Estimated Glomerular Filtration Rate for Live Kidney Donor Selection

Introduction

The assessment of the glomerular filtration rate (GFR) in kidney donor candidates is required for determining donor candidate acceptability. This assessment can be done using an estimated GFR (eGFR) or a measured GFR (mGFR). The primary objective of the present study was to compare, in healthy adult kidney donor candidates, GFR measured by the clearance of iothalamate to GFR estimated using the Chronic Kidney Disease Epidemiology Collaboration equation and to determine if eGFR was a suitable stand-alone assessment. A secondary objective was to explore demographic factors that affect the relationship of the eGFR and the mGFR.

Predictive Model for the Optimal Glomerular Filtration Rate in Living Kidney Transplant Recipients

Background

Recipient glomerular filtration rate (GFR) after living kidney transplantation (KT) is influenced by many factors. Defining the appropriate level of recipient GFR post-KT is helpful. The aim of this study was to establish a predictive model to estimate the optimal recipient GFR at 1 week post-KT.

Methods

We retrospectively analyzed 211 living KTs without delayed or slow graft function. Estimated GFR was calculated using the Cockcroft-Gault (CG) formula. Donor kidney volume was obtained from routine computed tomographic angiography (CTA) by work station GE (AW 4.20) program. Multivariate analysis was carried out with automated backward selection to establish the predictive model. The bias, precision, and accuracy of our model were also determined by application of the model to another 37 living KTs.

Results

In multivariate analysis, the significant parameters to predict recipient GFR were donor age (P = .025) and kidney volume (P < .0001) and both were incorporated in the predictive model; predicted CG recipient GFR = 28.325 + (donor kidney volume x 0.282) - (0.297 x donor age). The correlation coefficient (R) is 0.5. Application to another group revealed that our model had high precision (14.45 mL/min), small positive bias (0.24 mL/min), and high percentage (81%) of predicted value, which was within 30% of the observed recipient GFR post-KT.

Conclusion

Our predictive model included donor age and donor kidney volume and could be used to estimate the optimal recipient GFR post-KT. This could be helpful to identify early graft dysfunction and to make a decision if further invasive investigation such as allograft biopsy is necessary.     

Acute Effect of Furosemide on Glomerular Filtration Rate in Diastolic Dysfunction

We sought to evaluate the acute effect of furosemide on glomerular filtration rate (GFR) in subjects with diastolic dysfunction. An equal number of subjects with documented diastolic dysfunction (DD) and healthy volunteers (controls) were enrolled and underwent a baseline GFR measurement via plasma clearance of technetium-99m-diethylenetriaminepentaacetic acid. Within three to seven days of the baseline, study subjects were scheduled for a second GFR study, which was performed immediately after administration of furosemide (20 mg orally and 20 mg intravenously). There were eight healthy volunteers (8 males with a mean age 42 ± 7.8 years; 6 white, 2 Asian) and eight subjects with diastolic dysfunction (7 males, 1 female, with a mean age 64.5 ± 9.3 years; 7 whites, 1 African-American). There was a significant post-furosemide decline in GFR in the healthy volunteers, baseline vs. post-furosemide 131.6 ± 19.8 vs. 117 ± 18.2 mL/min, respectively (p?=?0.03), and the patients with DD, baseline vs. post-furosemide 117.5 ± 22.3 vs. 92 ± 21.7 mL/min, respectively (p?=?0.0002). A strong trend was detected, though not statistically significant, of greater GFR decline in subjects with DD compared to the healthy volunteers, 25.5 ± 9.9 vs. 14.6 ± 15.6 mL/min, respectively (p?=?0.12). To conclude, acute administration of furosemide might potentially cause a greater decline in GFR in subjects with diastolic dysfunction.