MO216PRELIMINARY STUDY OF THE GLYMPHATIC SYSTEM IN CKD Buonincontri, Veronica; Viggiano, Davide; Capasso, Giovambattista
Nephrology, dialysis, transplantation,
05/2021, Volume:
36, Issue:
Supplement_1
Journal Article
Peer reviewed
Abstract
Background and Aims
The glymphatic system is a network of extracellular spaces between neurons, glial cells, and capillaries that promotes the elimination of soluble molecules from the ...brain. Its dysfunction is probably relevant for neurodegenerative diseases such as Alzheimer's disease (AD). It is widely accepted that cognitive impairment accompanies chronic kidney disease (CKD). CKD is also a risk factor for dementia. However, the role of the glymphatic system in this process is unknown. A recent method to study the glymphatic system in human subjects has been proposed based on Diffusion Tensor Imaging (DTI) data and water diffusion calculation along with perivascular spaces. This approach is based on calculating a diffusion index named ALPS and showed that the glymphatic flow is reduced in MCI.
Method
To analyze the role of glymphatic system in CKD patients, we took advantage of the Alzheimer's Disease Neuroimaging Initiative (ADNI). ADNI is a longitudinal multicenter study helping researchers to monitor Alzheimer's disease and Mild Cognitive Impairment (MCI) progression. This database has a cohort of control patients and MCI patients, among which several patients with CKD stage II-III were identifiable from the creatinine values. Patients with Alzheimer's disease were excluded for this study. Among the control and MCI patients, we identified 12 CKD patients and pair-matched 12 non-CKD patients comparable for age, gender, and MoCA score. Magnetic resonance data with DTI sequences were retrieved for all patients, and the glymphatic system was characterized by the ALPS index. Tensor values were calculated using the FSL software; the diffusion values were calculated on tensor images using the ImageJ software. Differences in ALPS between CKD and non-CKD patients with and without MCI were tested.
Results
Analysis of DTI data confirmed that control patients without CKD had lower ALPS values when MCI was present compared to the non-MCI patients, suggesting a reduction of water diffusion in the glymphatic system. However, the presence of CKD had a different effect: in the absence of MCI, CKD did not modify ALPS values compared to non-CKD patients. At variance, in patients with MCI, CKD resulted in a significant increase of water diffusion in the glymphatic system compared to the controls.
Conclusion
In this preliminary study, MCI and CKD exerted opposite effects on the diffusion of water within the glymphatic system: MCI was accompanied by a reduction of water diffusion whereas CKD by an increased diffusion in the glymphatic spaces.
It is possible that small modification of water balance in CKD may be responsible for the increased diffusion of water in glymphatics in CKD. Further studies are needed to verify whether this unexpected phenomenon may modify cognitive function with a mechanism rather different from Alzheimer's disease.
KCNQ2 and KCNQ3 subunits encode for the muscarinic-regulated current (IKM), a sub-threshold voltage-dependent K⁺ current regulating neuronal excitability. In this study, we have investigated the ...involvement of IKM in dopamine (DA) release from rat striatal synaptosomes evoked by elevated extracellular K⁺ concentrations (K⁺e) and by muscarinic receptor activation. ³Hdopamine (³HDA) release triggered by 9 mmol/L K⁺e was inhibited by the IKM activator retigabine (0.01-30 μmol/L; Emax = 54.80 ± 3.85%; IC₅₀ = 0.50 ± 0.36 μmol/L). The IKM blockers tetraethylammonium (0.1-3 mmol/L) and XE-991 (0.1-30 μmol/L) enhanced K⁺-evoked ³HDA release and prevented retigabine-induced inhibition of depolarization-evoked ³HDA release. Retigabine-induced inhibition of K⁺-evoked ³HDA release was also abolished by synaptosomal entrapment of blocking anti-KCNQ2 polyclonal antibodies, an effect prevented by antibody pre-absorption with the KCNQ2 immunizing peptide. Furthermore, the cholinergic agonist oxotremorine (OXO) (1-300 μmol/L) potentiated 9 mmol/L K⁺e-evoked ³HDA release (Emax = 155 ± 9.50%; EC₅₀ = 25 ± 1.80 μmol/L). OXO (100 μmol/L)-induced ³HDA release enhancement was competitively inhibited by pirenzepine (1-10 nmol/L) and abolished by the M₃-preferring antagonist 4-diphenylacetoxy N-methylpiperidine methiodide (1 μmol/L), but was unaffected by the M₁-selective antagonist MT-7 (10-100 nmol/L) or by Pertussis toxin (1.5-3 μg/mL), which uncouples M₂- and M₄-mediated responses. Finally, OXO-induced potentiation of depolarization-induced ³HDA release was not additive to that produced by XE-991 (10 μmol/L), was unaffected by retigabine (10 μmol/L), and was abolished by synaptosomal entrapment of anti-KCNQ2 antibodies. Collectively, these findings indicate that, in rat striatal nerve endings, IKM channels containing KCNQ2 subunits regulate depolarization-induced DA release and that IKM suppression is involved in the reinforcement of depolarization-induced DA release triggered by the activation of pre-synaptic muscarinic heteroreceptors.
Abstract
Background and Aims
Treating renal diseases often requires ultrastructural details to establish or confirm diagnosis. Ultrastructural information that is not evidenced by optical microscopy ...includes, e.g., the podocyte foot processes effacement, thinning or alteration of basement membrane, presence of fibrils, Fabri's intracellular inclusions and subepithelial vs. subendothelial of deposits. Unfortunately, transmission electron microscopy (TEM), required to achieve the required resolution, has high costs, and is time-consuming. Here, we report our experience with scanning electron microscopy (SEM) on paraffin sections from standard pathology sections.
Method
We have retrospectively reanalyzed paraffin sections from renal biopsies of 50 patients with a diagnosis of IgAN, minimal change, diabetes, and amyloidosis. The paraffin sections were available and processed according to standard procedures (fixation, paraffin embedding), with a thickness of 3-4 µm. Sections were deparaffinized, stained with PASM (periodic acid Shiff + methenamine), mounted in aqueous mounting medium and imaged with 40x and 60x objectives. Sections were then unmounted, washed to remove glycerol, dehydrated in absolute alcohol, and air-dried. Finally, they were gold coated (“sputtering”) and analyzed in a scanning electron microscope (Zeiss).
Results
For the interpretation of SEM images, correlative analysis has been used, imaging precisely the same region of the section using standard optical microscopy and SEM. SEM can quickly identify the typical morphology of podocyte foot processes (Fig. 1A) and the podocyte effacement in minimal change disease (Fig. 1B). SEM can also easily allow the measurement of the thickness of the basement membrane of glomerular capillary walls (Fig. 1C-E) and its thinning in case of thin membrane disease (Fig. 1D) or its increased thickness in the case of diabetes (Fig. 1E). Amyloid fibrils (Fig. 1F) can also be identified in the capillary walls, confirming the diagnosis of amyloidosis Congo-red positive (Fig. 1G). The high resolution of the technique allows us to identify with high reliability the small ruptures of the capillary walls in IgA and membranoproliferative nephritis, with extravasation of erythrocytes and fibrin cloths (Fig. 1H). Mitochondria are well-demarcated, particularly in proximal tubule cells, and often, mitochondrial cristae are visible in samples with adequate fixation (Fig. 1I). Interstitial edema and fibrosis are evident in SEM microscopy (Fig. 1L). At very high magnification, in histological preparations with exceptional fixation, the filtration pores of endothelial cells could be observed (partially visible in Fig. 1E), as well as details such as the brush border of the tubular epithelial cells. The interpretation of the images is not always straightforward as it also requires knowledge of the artifacts induced by the fixation, processing, and inclusion of paraffin, such as, for example, the intracytoplasmic cellular structures containing lipids of the intratubular “foam cells” appear empty. For a correct interpretation of the SEM images, however, the recognition of the nuclei is essential because the chromatin is condensed into clods, especially around the periphery, and inside, some areas appear empty.
Conclusion
Scanning electron microscopy (SEM) is a powerful tool for the study of nephrological diseases, as it allows you to examine microscopic details at the nanometric scale (not detectable in optical microscopy), providing valuable information for diagnosis, understanding, and management of the disease, the possibility of carrying out Correlative Microscopy. Given the relatively simple and fast preparation of the samples to be examined, the low costs of execution, and the possibility of archiving the preparation in the histotheque for a possible revision of the case, this leads us to think that this method could be considered a valid alternative to transmission electron microscopy (TEM).
Abstract
Background and Aims
Congestive nephropathy is a kidney dysfunction caused by the impact of elevated venous pressures on renal hemodynamics and most of all it is due to right heart failure. ...Venous congestion can lead to a circulation vicious hormonal activation, increased intra-abdominal pressure, excessive renal tubular reabsorption of sodium and volume overload. In CKD with right heart failure, management of the patient is difficult because may be hard to identify a condition of fluid overload and when to stop intravenous diuretic treatment. Inferior caval vein (ICV) diameter and ICV index (reduction of diameter with respiration) have been used to identify fluid overload and guide diuretic treatment. However, in CKD patients with heart failure the ICV may not change rapidly and could overestimate the need of intravenous diuretics. This is why VExUS has been useful in both diagnosis and monitoring therapeutic. The VExUS method is a new approach, documented in the literature for the first time once in 2020. In this study we report the usefulness of the VExUS (Venous Excess Ultrasound Score) method for the diagnosis of overload volume and venous congestion and in setting an adequate therapy. It consists of Doppler evaluation of the abdominal veins: hepatic, portal and intrarenal. In fact, the acute renal failure, in the patients studied, is to be attributed in the first instance to venous congestion rather than underfilling, resulting from decompensation and therefore requiring diuretic therapy.
Method
This retrospective study was conducted in CKD patients (stages 3-4) with acute kidney injury (creatinine level: 6.3-2.6 mg/dl) due to right heart failure admitted at the U.O.C. Nephrology at the S. Maria della Pietà Hospital in Nola and UOC Nephrology of Univ. of Campania. The VExUS method defines 3 degrees (score 0-3) of congestion based on the severity of the anomalies on hepatic, portal and intrarenal venous Doppler. All patients have been treated with diuretics i.v. based on a VEXUS score>1, and then returned to oral treatment when VEXUS score returned to 0. VExUS assessment was performed at admission, repeated every 48 hours and the last score has been considered here (on average 1 week). The relationship between change in VExUS score and ICV index vs creatinine levels was studied by correlation analysis. Lung POCUS (point of care pulmonary ultrasonography) has also been used for all patients to identify a possible contemporary overload caused by LV failure.
Results
Thirty patients have been included. The use of diuretics reduced the VExUS score and the ICV diameter. However, diuretic treatment changed the ICV diameter by a smaller extent (−30%) compared to the VExUS score (−80%), and one patient showed clear reduction of VExUS score, without any modification of the ICV diameter, suggesting that the latter is a slower indicator of recovery compared to VExUS. Indeed, the change of VExUS score was highly correlated to the improvement of creatinine levels (Pearson's correlation 0.837, p < 0.001). The change of IVC diameter explained in comparison a smaller amount of variability of the creatinine improvement (Pearson's correlation 0.65, p = 0.008).
Conclusion
VExUS is a promising and reliable tool in the management of congestive nephropathy in patients with CKD and right heart failure. VExUS is superior to VCI diameter and should be used routinely, along with clinical, laboratory, and other ultrasound parameters, because of its usefulness, repeatability, low cost and ease of execution.
Abstract
Background and Aims
The sodium-glucose co-transporter type 2 inhibitors (SGLT2i) such as dapagliflozin and empagliflozin, are a new class of nephro- and cardio-protective drugs in diabetic ...and non-diabetic patients. SGLT2 is greatly expressed in the proximal tubule cells, and therefore the protective effects of gliflozins are thought to be mediated by a hemodynamic mechanism on glomerular hyperfiltration. However, they could be acting also in other cell types, particularly in diabetic patients, because hyperglycemia induces the expression of SGLT2 in endothelial cells, mesangial cells, cardiomyocytes, etc. Because SGLT2 changes the intracellular concentration of sodium and glucose, gliflozins may indirectly change mitochondrial swelling and activity. In this study, we have tested the hypothesis that gliflozins act on mitochondria in patients suffering from diabetes.
Method
We have analyzed kidney biopsies from diabetic patients in treatment with a gliflozin (n = 10) or without gliflozin treatment (n = 10) stained with fuchsin red, which gives a color intensity proportional to mitochondrial content. Clinical variables (albumin-creatinine ratio (ACR), blood pressure, eGFR, glycated albumin) have been measured before and after 4 months of treatment. Images have been acquired using a Zeiss Axioscope with 100x objective and analyzed using ImageJ software to quantify staining intensity (expressed as optical density) after background subtraction. Two different cell types have been considered: proximal tubule cells and endothelial cells in glomeruli. Only non-sclerotic glomeruli have been considered.
Results
The systolic blood pressure was linearly correlated to the intensity of staining of mitochondria in the proximal tubule cells in all patients irrespective of gliflozin treatment. In non-treated diabetic patients, the mitochondrial content of endothelial cells was not related to the extent of ACR reduction over time. In diabetic patients treated with gliflozins, the extent of ACR reduction after treatment was correlated with the intensity of mitochondrial staining in glomerular endothelial cells. This correlation was not present in proximal tubule cells. Gliflozin treatment may induce a change in the mitochondrial content of endothelial cells, particularly in patients responding with a reduction in proteinuria.
Conclusion
Proteinuria in diabetic patients depends on damage in both the endothelial and podocyte compartments. Gliflozins may improve proteinuria by a hemodynamic mechanism; however, our data suggest an effect of gliflozins specific on mitochondria of the endothelial cells in the glomeruli, which is proportional to their anti-proteinuric effect. Though we do not analyze the mechanism of gliflozins on mitochondria, it is possible to speculate that the changes of intracellular sodium-induced by gliflozins may induce an altered mitochondrial activity.
Abstract
Background and Aims
Autosomal Dominant Tubulointerstitial kidney disease (ADTKD) is a rare disease (16 per million), associated with CV events, hypertension, and gout. ADTKD is characterized ...by interstitial fibrosis, tubular atrophy, microcysts, and progressive CKD. We describe a case of a family with clinical characteristics of ADTKD and provide for the first time a characterization of their urinary sediment. Given the tendency of a specific type of ADTKD caused by UMOD mutation to form tubular crystals, we have also tested the ability of the urine from ADTKD patients to precipitate and form crystals in vitro.
Method
We examined clinical features and urinary sediment of a family with ADTKD: a 51 years old woman with CKD (eGFR 30 mL/min/1.73 m2, CDK-EPI 2021) and her three sons, a 20 years old male, two daughters of 26 and 24 years old, all with hyperuricemia. Genetic analysis is not yet available. The test of urine precipitation consisted of the analysis of crystals after air-drying a thin layer of urine after enrichment of the organic quota by triple centrifugation for 10 minutes at 1000 rpm. Urinary sediments were also studied by Atomic Force Microscopy (ICSPI nGauge AFM) to achieve a resolution of 20 nm of crystals.
Results
Clinical data for the four patients are reported in the table. None of the patients showed proteinuria.
The urine sediment of all patients showed rare WBC (in greater amounts only in patient 1), rare amorphous granular casts, and rare tubular cells. Three patients showed formations of variably sized crystals in vitro.
The precipitation test did not show crystals in patient 1, whereas patients 2-3 showed several rosette-type crystals often with a wagon-wheel concentric contour which resembled leucine crystals or CaCO3. Adding formalin to the samples resulted in a more clear organization of crystals in wagon-wheel structures. AFM imaging of the crystals showed rod-like structures branching with an angle of 35°.
Conclusion
ADTKD remains a poorly understood genetic condition with no specific treatment options. Our data suggest that patients with this condition may have an enhanced tendency to form crystals in vitro. Speculatively, this might induce tubular injury in vivo. Data from UMOD-ko animal models showed intrarenal crystals (not better specified). Genetic alterations of branched aminoacids metabolism (such as leucine in Maple syrup disease) can also cause tubular diseases, although the systemic manifestations are very different from our patients. Genetic analysis of our family will give further information on the proteins involved in these crystal structures.
Abstract
Background and Aims
Mild Cognitive Impairment (MCI) is a common finding in chronic kidney disease (CKD) patients. Indeed, CKD represents a relevant risk factor for developing dementia and ...MCI. Cholinesterase inhibitors, such as rivastigmine, are among the few drugs approved for the treatment of dementia and MCI. Rivastigmine is also used to treat vascular dementia because it protects subcortical brain structures. Data are scanty regarding the use of rivastigmine in CKD patients with MCI and are much needed to guide the therapy for MCI in this cohort of patients.
Method
This retrospective case-control study compared the effects of rivastigmine on cognitive functions in MCI patients with CKD (stage III-IV; n= 20) and without CKD (n=21, control group), comparable for the extent of cognitive impairment (indexed by Montreal Cognitive Assessment, MoCA), age (range 18-65 years), gender, weight, and comorbidities. Patients under treatment with rivastigmine and with a baseline MoCA score available were included in the study. Exclusion criteria were ictus, psychiatric or other neurological conditions, heart failure, liver failure, severe obesity, anemia, electrolyte disorders, cancer, dialysis, and other severe comorbidities. Laboratory test data (glycemia, cholesterol, hemoglobin, proteinuria, creatinine) were used to characterize the two populations. MCI was defined as a MoCA score between 21-26. The cognitive screening was available at baseline (before treatment) and during a follow-up in a range of three-six months after the start of the treatment. CKD was defined by eGFR < 60 mL/min/1.73m2.
Results
The follow-up timing for cognitive screening was not statistically different between the two cohorts. The control group (MCI without CKD) showed a small, significant improvement in the MoCA score after treatment (baseline MoCA: 22.9±0.5, follow-up MoCA: 23.5±0.5, p=0.02, t-test for paired data).
At variance, the MCI-CKD group showed a significant improvement in the MoCA score (baseline MoCA=23±0.4, follow-up MoCA=24.3±0.4, p<0.05).
Accordingly, the extent of improvement of MoCA score after rivastigmine was inversely correlated to the eGFR (r = -0.23).
Conclusion
A significant improvement in MoCA score accompanied treatment with rivastigmine in the CKD group. More extensive population studies are needed to verify the greater efficacy of Acetylcholinesterase inhibitors in this population.
Purpose
The purpose of this study was to report the clinical and functional results of a series of patients with early knee osteoarthritis (KOA) treated with the intra-articular injection of ...autologous adipose-derived stem cells (aASCs) plus arthroscopic debridement. The hypothesis was that protocol would significantly improve the clinical and functional outcomes in patients with early KOA.
Methods
Fifty-two patients with early KOA, who received arthroscopic debridement followed by percutaneous injection of aASCs, were enrolled into the study and retrospectively analyzed with an average follow-up of 15.3 (range, 6 to 24) months. Patients were assessed through the IKS knee and function scores and VAS pain scale.
Results
The mean IKS knee score improved from 37.4 (range, 14 to 79) points pre-operatively to 62.6 (range, 27 to 95) points at the latest follow-up (
p
< < 0.01). The mean IKS function score improved from 57.2 (range, 25 to 100) points pre-operatively to 83.0 (range, 35 to 100) points at the latest follow-up (
p
< < 0.01). The mean VAS score decreased from 8.5 (range, 3 to 10) pre-operatively to 5.1 (range, 0 to 8) at the latest follow-up (
p
< < 0.01). Additionally, patients with a pre-operative VAS score greater than 8 were found to show greater clinical and functional benefits compared with patients with VAS score lower than 8.
Conclusions
The knee injection of aASCs associated to arthroscopic debridement increased significantly the clinical and functional scores in patients with early KOA at a mid-term follow-up, especially those with higher pre-operative VAS scores.
Abstract
Background and Aims
Standard ultrasound mainly provides information about structural changes in the kidney, whereas parenchymal Doppler ultrasound provides the renal resistance index (RI), ...which correlates with plasma creatinine levels. Unfortunately, in the case of isolated proteinuria and normal renal function/creatinine levels, these two classical approaches do not show any changes. Recent advances in ultrasound analysis, allowing the measure of kidney stiffness (sharewave elastometry) and the imaging of ultralow-flow vessels up glomeruli (such as X-Flow), might be helpful to identify kidney alterations in case of proteinuria with normal creatinine.
We hypothesize that proteinuria with intact glomerular filtration may induce a measurable change in kidney stiffness and the size of low-flow microcircle, with different effects in acute and chronic conditions. We also verify whether the mismatch between creatinine, resistance index, and kidney size may suggest a specific nephrological disease.
Method
We conducted a transversal study on 301 patients with chronic kidney disease (CKD) and 160 kidney transplant patients (Tx), comparable for age, gender, and eGFR. For all patients, we retrieved the resistance indices of both the kidney and the spleen, clinical variables, shear wave elastrometry, and the X-Flow algorithm (Esaote) to detect the microcircle. We compared Conventional pulsed wave Doppler techniques (resistance index, RI) and ultrasensitive Doppler algorithm (Xflow, Esaote, Genoa, GE, Italy) to study the perfusion pattern and smaller angio-architecture of kidney's cortical vessels. X-Flow is a high-resolution and high-definition Doppler technology, which, with a new algorithm, makes better spatial resolution possible and maximizes Doppler signal penetration capabilities. We also identify a mismatch between creatinine and the renal RI, calculating the renal mismatch index (correlation between renal resistance index and creatinine level). The connection between the resistance index and the kidney size was tested as a significant marker of chronicity.
Results
The eGFR was best predicted by renal RI in Tx patients (Pearson −0.4, p < 0.01). At variance, the RI is a poor predictor of eGFR in CKD patients: in these subjects, the kidney longitudinal size was a better predictor of eGFR (Pearson's coefficient 0.39, p < 0.01). Using a large dataset from a previous study, we estimated the total number of nephrons using the following approximation: nephron number = (5270*total cortical volume – 61612)*number of kidneys. This formula is only valid for chronic patients and cannot be applied to polycystic patients. We speculated that while eGFR was proportional to the number of active glomeruli in CKD patients (thus correlating with kidney size), in TX patients, the local microcircle better explained the changes in eGFR. The consequence is that the eGFR has different meaning in CKD and Tx patients.
Patients deviating from this pattern are identified as having an increased renal dissimilarity index. The multiple regression analysis revealed that RI also depended on age and cholesterol levels. The spleen resistance index correlated with cholesterol levels. The kidney mismatch index defined a subgroup of patients with tubular microcirculation abnormalities. Shearwave stiffness correlated with Resistance index (Pearson −0.3, p = 0.04) and, limited to Tx patients, to the cortical volume (Pearson = −0.446, p < 0.05) and number of nephrons (Pearson −0.44, p < 0.05), but was not associated with creatinine or eGFR. The extent of microcircle measured on X-flow images was altered in chronic forms of proteinuria with normal creatinine levels.
Conclusion
Kidney elastometry and kidney X-flow indices might show more sources of profitable information that can be collected from ultrasound approaches that allow the identification of a diseased kidney, even in the presence of regular architecture and average resistance index. The X-flow algorithm should be used to study the smaller vascular tree not visualizable with Doppler, such as the afferent arteriole.
The hyperfunctioning dopamine hypothesis in the mesocorticolimbic (MCL) system has been addressed by a neurogenetic approach in model systems. Thus, a morphometric analysis was carried out on neurons ...of origin of Substantia Nigra (SN) and Ventral Tegmental Area (VTA) dopamine systems of the Naples High-Excitability (NHE), Low-Excitability (NLE) and control lines. Male adult rats were tested in a spatial novelty for indices of activity and non-selective attention. Mesencephalic coronal sections were processed for tyrosine hydroxylase (TH) immunohistochemistry and cytochromoxidase (C.O.) histochemistry. Image analysis in the rostro-caudal plane showed (i) a higher neuron size of TH+ elements in the VTA of NHE and NLE, across the entire structure in the NHE, and only in the middle portion in the NLE; (ii) a higher expression of TH in the neuropil of the VTA in NHE; (iii) a lower C.O. activity in both NLE and NHE; (iv) no differences in the SN. The larger neuron size in both NHE and NLE rats as compared with control rats, along with higher TH expression mainly in the NHE, in absence of any relevant alteration in the SN, reveals an unbalance between the two dopamine systems and a subsequent alteration in limbic (reward, motivation, sustained attention) functions. The decreased C.O. activity might be due to reduced feedback inhibition by striatal GABA neurons and interneurons leading to increased DA neuron firing. In conclusion, the increased behavioral activity and impaired attention observed in the NHE rats are associated to hyperfunctioning MCL system in this genetic model of Attention-Deficit Hyperactivity Disorder (ADHD).
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