Low Salt Delivery Triggers Autocrine Release of Prostaglandin E2 From the Aldosterone-Sensitive Distal Nephron in Familial Hyperkalemic Hypertension Mice

Aberrant activation of with-no-lysine kinase (WNK)-STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) kinase signaling in the distal convoluted tubule (DCT) causes unbridled activation of the thiazide-sensitive sodium chloride cotransporter (NCC), leading to familial hyperkalemic hypertension (FHHt) in humans. Studies in FHHt mice engineered to constitutively activate SPAK specifically in the DCT (CA-SPAK mice) revealed maladaptive remodeling of the aldosterone sensitive distal nephron (ASDN), characterized by decrease in the potassium excretory channel, renal outer medullary potassium (ROMK), and epithelial sodium channel (ENaC), that contributes to the hyperkalemia. The mechanisms by which NCC activation in DCT promotes remodeling of connecting tubule (CNT) are unknown, but paracrine communication and reduced salt delivery to the ASDN have been suspected. Here, we explore the involvement of prostaglandin E2 (PGE2). We found that PGE2 and the terminal PGE2 synthase, mPGES1, are increased in kidney cortex of CA-SPAK mice, compared to control or SPAK KO mice. Hydrochlorothiazide (HCTZ) reduced PGE2 to control levels, indicating increased PGE2 synthesis is dependent on increased NCC activity. Immunolocalization studies revealed mPGES1 is selectively increased in the CNT of CA-SPAK mice, implicating low salt-delivery to ASDN as the trigger. Salt titration studies in an in vitro ASDN cell model, mouse CCD cell (mCCD-CL1), confirmed PGE2 synthesis is activated by low salt, and revealed that response is paralleled by induction of mPGES1 gene expression. Finally, inhibition of the PGE2 receptor, EP1, in CA-SPAK mice partially restored potassium homeostasis as it partially rescued ROMK protein abundance, but not ENaC. Together, these data indicate low sodium delivery to the ASDN activates PGE2 synthesis and this inhibits ROMK through autocrine activation of the EP1 receptor. These findings provide new insights into the mechanism by which activation of sodium transport in the DCT causes remodeling of the ASDN.

Automated Segmentation of Kidney Cortex and Medulla in CT Images: A Multisite Evaluation Study

Background: In kidney transplantation, a contrast CT scan is obtained in the candidate kidney donor to detect any subclinical pathology in the kidney. Recent work from the Aging Kidney Anatomy study has characterized kidney volumes, cortex volumes, and medulla volumes using a manual image processing tool. However, this technique is time consuming and impractical for clinical care, and thus, these measures are not obtained during donor evaluations. This study proposes a fully automated segmentation approach for measuring kidney, cortex, and medulla volumes. Methods: A total of 1930 contrast-enhanced CT exams with reference standard manual segmentations from one institution were used for development of the algorithm. A convolutional neural network model was trained (n=1238) and validated (n=306), and then evaluated in a hold-out test set of reference standard segmentations (n=386). After the initial evaluation, the algorithm was further tested on datasets originating from two external sites (n=1226). Results: The automated model was found to perform on par with manual segmentation with errors similar to interobserver variability with manual segmentations. Compared with the reference standard, the automated approach achieved a Dice similarity metric of 0.94 (right cortex), 0.90 (right medulla), 0.94 (left cortex), and 0.90 (left medulla) in the test set. Similar performance was observed when the algorithm was applied on the two external datasets. Conclusions: A fully automated approach for measuring cortex and medullary volumes in CT images of the kidneys has been established. This method may prove useful for a wide range of clinical applications.

Neuron-like function of the nephron central command

SUMMARYInteroceptive neurons that sense and regulate our internal milieu have been identified in several organs except in the kidney cortex despite its major importance in maintaining body homeostasis. Here we report that the chief kidney cell type of the macula densa (MD) forms coordinated neural networks in each nephron that resemble peripheral ganglia. A combined in vivo single-cell 4D physiology (sc4DP) and scRNA sequencing approach identified the MD mechanisms of neuronal differentiation, heterogeneity (pacemaker MD cells), sensing of the local and systemic environment via multi-organ crosstalk, and regulation of organ functions by acting as the nephron central command. Consistent with their neuron-like nature, MD cells express the molecular fingerprint of neurodegeneration. Here we put forth the single-cell MD model and concept of local neural networks that control organ and body functions via interoception in normal physiological state and use an integrated mechanism of neurodegeneration in disease.

Systems biology and machine learning approaches identify drug targets in diabetic nephropathy

Diabetic nephropathy (DN), the leading cause of end-stage renal disease, has become a massive global health burden. Despite considerable efforts, the underlying mechanisms have not yet been comprehensively understood. In this study, a systematic approach was utilized to identify the microRNA signature in DN and to introduce novel drug targets (DTs) in DN. Using microarray profiling followed by qPCR confirmation, 13 and 6 differentially expressed (DE) microRNAs were identified in the kidney cortex and medulla, respectively. The microRNA-target interaction networks for each anatomical compartment were constructed and central nodes were identified. Moreover, enrichment analysis was performed to identify key signaling pathways. To develop a strategy for DT prediction, the human proteome was annotated with 65 biochemical characteristics and 23 network topology parameters. Furthermore, all proteins targeted by at least one FDA-approved drug were identified. Next, mGMDH-AFS, a high-performance machine learning algorithm capable of tolerating massive imbalanced size of the classes, was developed to classify DT and non-DT proteins. The sensitivity, specificity, accuracy, and precision of the proposed method were 90%, 86%, 88%, and 89%, respectively. Moreover, it significantly outperformed the state-of-the-art (P-value ≤ 0.05) and showed very good diagnostic accuracy and high agreement between predicted and observed class labels. The cortex and medulla networks were then analyzed with this validated machine to identify potential DTs. Among the high-rank DT candidates are Egfr, Prkce, clic5, Kit, and Agtr1a which is a current well-known target in DN. In conclusion, a combination of experimental and computational approaches was exploited to provide a holistic insight into the disorder for introducing novel therapeutic targets.

Tempol Alters Urinary Extracellular Vesicle Lipid Content and Release While Reducing Blood Pressure during the Development of Salt-Sensitive Hypertension

Salt-sensitive hypertension resulting from an increase in blood pressure after high dietary salt intake is associated with an increase in the production of reactive oxygen species (ROS). ROS are known to increase the activity of the epithelial sodium channel (ENaC), and therefore, they have an indirect effect on sodium retention and increasing blood pressure. Extracellular vesicles (EVs) carry various molecules including proteins, microRNAs, and lipids and play a role in intercellular communication and intracellular signaling in health and disease. We investigated changes in EV lipids, urinary electrolytes, osmolality, blood pressure, and expression of renal ENaC and its adaptor protein, MARCKS/MARCKS Like Protein 1 (MLP1) after administration of the antioxidant Tempol in salt-sensitive hypertensive 129Sv mice. Our results show Tempol infusion reduces systolic blood pressure and protein expression of the alpha subunit of ENaC and MARCKS in the kidney cortex of hypertensive 129Sv mice. Our lipidomic data show an enrichment of diacylglycerols and monoacylglycerols and reduction in ceramides, dihydroceramides, and triacylglycerols in urinary EVs from these mice after Tempol treatment. These data will provide insight into our understanding of mechanisms involving strategies aimed to inhibit ROS to alleviate salt-sensitive hypertension.

Effects of Renal Denervation on the Enhanced Renal Vascular Responsiveness to Angiotensin II in High-Output Heart Failure: Angiotensin II Receptor Binding Assessment and Functional Studies in Ren-2 Transgenic Hypertensive Rats

Detailed mechanism(s) of the beneficial effects of renal denervation (RDN) on the course of heart failure (HF) remain unclear. The study aimed to evaluate renal vascular responsiveness to angiotensin II (ANG II) and to characterize ANG II type 1 (AT1) and type 2 (AT2) receptors in the kidney of Ren-2 transgenic rats (TGR), a model of ANG II-dependent hypertension. HF was induced by volume overload using aorto-caval fistula (ACF). The studies were performed two weeks after RDN (three weeks after the creation of ACF), i.e., when non-denervated ACF TGR enter the decompensation phase of HF whereas those after RDN are still in the compensation phase. We found that ACF TGR showed lower renal blood flow (RBF) and its exaggerated response to intrarenal ANG II (8 ng); RDN further augmented this responsiveness. We found that all ANG II receptors in the kidney cortex were of the AT1 subtype. ANG II receptor binding characteristics in the renal cortex did not significantly differ between experimental groups, hence AT1 alterations are not responsible for renal vascular hyperresponsiveness to ANG II in ACF TGR, denervated or not. In conclusion, maintained renal AT1 receptor binding combined with elevated ANG II levels and renal vascular hyperresponsiveness to ANG II in ACF TGR influence renal hemodynamics and tubular reabsorption and lead to renal dysfunction in the high-output HF model. Since RDN did not attenuate the RBF decrease and enhanced renal vascular responsiveness to ANG II, the beneficial actions of RDN on HF-related mortality are probably not dominantly mediated by renal mechanism(s).

The Extracellular Matrix Environment of Clear Cell Renal Cell Carcinoma Determines Cancer Associated Fibroblast Growth

Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer and is often caused by mutations in the oxygen-sensing machinery of kidney epithelial cells. Due to its pseudo-hypoxic state, ccRCC recruits extensive vasculature and other stromal components. Conventional cell culture methods provide poor representation of stromal cell types in primary cultures of ccRCC, and we hypothesized that mimicking the extracellular environment of the tumor would promote growth of both tumor and stromal cells. We employed proteomics to identify the components of ccRCC extracellular matrix (ECM) and found that in contrast to healthy kidney cortex, laminin, collagen IV, and entactin/nidogen are minor contributors. Instead, the ccRCC ECM is composed largely of collagen VI, fibronectin, and tenascin C. Analysis of single cell expression data indicates that cancer-associated fibroblasts are a major source of tumor ECM production. Tumor cells as well as stromal cells bind efficiently to a nine-component ECM blend characteristic of ccRCC. Primary patient-derived tumor cells bind the nine-component blend efficiently, allowing to us to establish mixed primary cultures of tumor cells and stromal cells. These miniature patient-specific replicas are conducive to microscopy and can be used to analyze interactions between cells in a model tumor microenvironment.

Lipidomics Reveals Cisplatin-Induced Renal Lipid Alterations during Acute Kidney Injury and Their Attenuation by Cilastatin

Nephrotoxicity is a major complication of cisplatin-based chemotherapy, leading to acute kidney injury in ca. 30% of patients, with no preventive intervention or treatment available for clinical use. Cilastatin has proved to exert a nephroprotective effect for cisplatin therapies in in vitro and in vivo models, having recently entered clinical trials. A deeper understanding at the molecular level of cisplatin-induced renal damage and the effect of potential protective agents could be key to develop successful nephroprotective therapies and to establish new biomarkers of renal damage and nephroprotection. A targeted lipidomics approach, using LC-MS/MS, was employed for the quantification of 108 lipid species (comprising phospholipids, sphingolipids, and free and esterified cholesterol) in kidney cortex and medulla extracts from rats treated with cisplatin and/or cilastatin. Up to 56 and 63 lipid species were found to be altered in the cortex and medulla, respectively, after cisplatin treatment. Co-treatment with cilastatin attenuated many of these lipid changes, either totally or partially with respect to control levels. Multivariate analysis revealed that lipid species can be used to discriminate renal damage and nephroprotection, with cholesterol esters being the most discriminating species, along with sulfatides and phospholipids. Potential diagnostic biomarkers of cisplatin-induced renal damage and cilastatin nephroprotection were also found.

Comprehensive Evaluation of Caloric Restriction-Induced Changes In The Metabolome Profile of Mice

Objects: Caloric restriction (CR) is known to extend lifespan and exert a protective effect on organs, and is thus a low-cost and easily implemented approach to the health maintenance. However, there have been no studies that have systematically evaluated the metabolic changes that occur in the main tissues affected by CR. This study aimed to explore the target tissues metabolomic profile in CR mice.Methods: Male C57BL/6J mice were randomly allocated to the CR group (n = 7) and control group (n = 7). A non-targeted gas chromatography–mass spectrometry (GC–MS) approach and multivariate analysis were used to identify metabolites in the main tissues (serum, heart, liver, kidney, cortex, hippocampus, lung, muscle, and white adipose) in model of CR. Results: We identified 10 metabolites in the heart that showed differential abundance between the 2 groups, along with 9 in kidney, 7 in liver, 6 in lung, 6 in white adipose, 4 in hippocampus, 4 in serum, 3 in cortex, and 2 in muscle. The most significantly altered metabolites were amino acids (AAs) (glycine, aspartic acid, l-isoleucine, l-proline, l-aspartic acid, l-serine, l-hydroxyproline, l-alanine, l-valine, l-threonine, l-glutamic acid, and phenylalanine) and fatty acids (FAs) (palmitic acid, 1-monopalmitin, glycerol monostearate, docosahexaenoic acid, 16-octadecenoic acid, oleic acid, stearic acid, and hexanoic acid). These metabolites were associated with 8 different functional pathways related to the metabolism of AAs, lipids, and energy. Conclusion: Our results provide insight into the specific metabolic changes that are induced by CR and can serve as a reference for physiologic studies on how CR improves health and extends lifespan.