Ghrelin enhances tubular magnesium absorption in the kidney.

Osteoporosis after bariatric surgery is an increasing health concern as the rate of bariatric surgery has risen. In animal studies mimicking bariatric procedures, bone disease, together with decreased serum levels of Ca2+, Mg2+ and the gastric hormone Ghrelin were described. Ghrelin regulates metabolism by binding to and activating the growth hormone secretagogue receptor (GHSR) which is also expressed in the kidney. As calcium and magnesium are key components of bone, we tested the hypothesis that Ghrelin-deficiency contributes to osteoporosis via reduced upregulation of the renal calcium channel TRPV5 and the heteromeric magnesium channel TRPM6/7. We expressed GHSR with TRPV5 or TRPM6/7 channel in HEK293 cells and treated them with purified Ghrelin. Whole-cell current density was analyzed by patch-clamp recording. Nephron-specific gene expression was performed by tubular microdissection followed by qPCR in wild-type (WT) mice, and immunofluorescent imaging of GHSR-eGFP mice. Tubular magnesium homeostasis was analyzed in GHSR-null and WT mice at baseline and after caloric restriction. After Ghrelin exposure, whole-cell current density did not change for TRPV5 but increased for TRPM6/7 in a dose-dependent fashion. Applying the Ghrelin-mimetic (D-Trp7, Ala8,D-Phe10)-α-MSH (6-11) amide without and with the GHSR antagonist (D-Lys3)-GHRP6, we confirmed the stimulatory role of Ghrelin towards TRPM6/7. As GHSR initiates downstream signaling via protein kinase A (PKA), we found that the PKA inhibitor H89 abrogated TRPM6/7 stimulation by Ghrelin. Similarly, transfected Gαs, but not the Gαs mutant Q227L, nor Gαi2, Gαq, or Gα13 upregulated TRPM6/7 current density. In microdissected TALs and DCTs similar levels of GHSR mRNA were detected. In contrast, TRPM6 mRNA was expressed in the DCT and also detected in the TAL at 25% expression compared to DCT. Immunofluorescent studies using reporter GHSR-eGFP mice showed a strong eGFP signal in the TAL but surprisingly displayed no eGFP signal in the DCT. In 3-, 6-, and 9-month-old GHSR-null and WT mice, baseline serum magnesium was not significantly different, but 24-h urinary magnesium excretion was elevated in 9-month-old GHSR-null mice. In calorically restricted GHSR-null mice, we detected excess urinary magnesium excretion and reduced serum magnesium levels compared to WT mice. The kidneys from calorically restricted WT mice showed upregulated gene expression of magnesiotropic genes Hnf1b, Cldn-16, Cldn-19, Fxyd-2b, and Parvalbumin compared to GHSR-null mice. Our in vitro studies show that Ghrelin stimulates TRPM6/7 via GHSR and Gαs-PKA signaling. The murine studies are consistent with Ghrelin-GHSR signaling inducing reduced urinary magnesium excretion, particularly in calorically restricted mice when Ghrelin levels are elevated. This effect may be mediated by Ghrelin-upregulation of TRPM6 in the TAL and/or upregulation of other magnesiotropic genes. We postulate that rising Ghrelin levels with hunger contribute to increased renal Mg2+ reabsorption to compensate for lack of enteral Mg2+ uptake.

Rap1 regulates lumen continuity via Afadin in renal epithelia.

The continuity of a lumen within an epithelial tubule is critical for its function. We previously found that the F-actin binding protein Afadin is required for timely lumen formation and continuity in renal tubules formed from the nephrogenic mesenchyme in mice. Afadin is a known effector and interactor of the small GTPase Rap1, and in the current study, we examine the role of Rap1 in nephron tubulogenesis. Here, we demonstrate that Rap1 is required for nascent lumen formation and continuity in cultured 3D epithelial spheroids and in vivo in murine renal epithelial tubules derived from the nephrogenic mesenchyme, where its absence ultimately leads to severe morphogenetic defects in the tubules. By contrast, Rap1 is not required for lumen continuity or morphogenesis in renal tubules derived from the ureteric epithelium, which differ in that they form by extension from a pre-existing tubule. We further demonstrate that Rap1 is required for correct localization of Afadin to adherens junctions both in vitro and in vivo. Together, these results suggest a model in which Rap1 localizes Afadin to junctional complexes, which in turn regulates nascent lumen formation and positioning to ensure continuous tubulogenesis.

Heterozygous Mutation of Vegfr3 Reduces Renal Lymphatics without Renal Dysfunction.

BACKGROUND: Lymphatic abnormalities are observed in several types of kidney disease, but the relationship between the renal lymphatic system and renal function is unclear. The discovery of lymphatic-specific proteins, advances in microscopy, and available genetic mouse models provide the tools to help elucidate the role of renal lymphatics in physiology and disease. METHODS: We utilized a mouse model containing a missense mutation in Vegfr3 (dubbed Chy ) that abrogates its kinase ability. Vegfr3 Chy/+ mice were examined for developmental abnormalities and kidney-specific outcomes. Control and Vegfr3 Chy/+ mice were subjected to cisplatin-mediated injury. We characterized renal lymphatics using tissue-clearing, light-sheet microscopy, and computational analyses. RESULTS: In the kidney, VEGFR3 is expressed not only in lymphatic vessels but also, in various blood capillaries. Vegfr3 Chy/+ mice had severely reduced renal lymphatics with 100% penetrance, but we found no abnormalities in BP, serum creatinine, BUN, albuminuria, and histology. There was no difference in the degree of renal injury after low-dose cisplatin (5 mg/kg), although Vegfr3 Chy/+ mice developed perivascular inflammation. Cisplatin-treated controls had no difference in total cortical lymphatic volume and length but showed increased lymphatic density due to decreased cortical volume. CONCLUSIONS: We demonstrate that VEGFR3 is required for development of renal lymphatics. Our studies reveal that reduced lymphatic density does not impair renal function at baseline and induces only modest histologic changes after mild injury. We introduce a novel quantification method to evaluate renal lymphatics in 3D and demonstrate that accurate measurement of lymphatic density in CKD requires assessment of changes to cortical volume.

Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution.

We present cleared-tissue axially swept light-sheet microscopy (ctASLM), which enables isotropic, subcellular resolution imaging with high optical sectioning capability and a large field of view over a broad range of immersion media. ctASLM can image live, expanded, and both aqueous and non-aqueous chemically cleared tissue preparations. Depending on the optical configuration, ctASLM provides up to 260 nm of axial resolution, a three to tenfold improvement over confocal and other reported cleared-tissue light-sheet microscopes. We imaged millimeter-scale cleared tissues with subcellular three-dimensional resolution, which enabled automated detection of multicellular tissue architectures, individual cells, synaptic spines and rare cell-cell interactions.

Nephronectin Regulates Mesangial Cell Adhesion and Behavior in Glomeruli.

A critical aspect of kidney function occurs at the glomerulus, the capillary network that filters the blood. The glomerular basement membrane (GBM) is a key component of filtration, yet our understanding of GBM interactions with mesangial cells, specialized pericytes that provide structural stability to glomeruli, is limited. We investigated the role of nephronectin (Npnt), a GBM component and known ligand of α8ß1 integrin. Immunolocalization and in situ hybridization studies in kidneys of adult mice revealed that nephronectin is produced by podocytes and deposited into the GBM. Conditional deletion of Npnt from nephron progenitors caused a pronounced increase in mesangial cell number and mesangial sclerosis. Nephronectin colocalized with α8ß1 integrin to novel, specialized adhesion structures that occurred at sites of mesangial cell protrusion at the base of the capillary loops. Absence of nephronectin disrupted these adhesion structures, leading to mislocalization of α8ß1. Podocyte-specific deletion of Npnt also led to mesangial sclerosis in mice. These results demonstrate a novel role for nephronectin and α8ß1 integrin in a newly described adhesion complex and begin to uncover the molecular interactions between the GBM and mesangial cells, which govern mesangial cell behavior and may have a role in pathologic states.

Afadin orients cell division to position the tubule lumen in developing renal tubules.

In many types of tubules, continuity of the lumen is paramount to tubular function, yet how tubules generate lumen continuity in vivo is not known. We recently found that the F-actin-binding protein afadin is required for lumen continuity in developing renal tubules, though its mechanism of action remains unknown. Here, we demonstrate that afadin is required for lumen continuity by orienting the mitotic spindle during cell division. Using an in vitro 3D cyst model, we find that afadin localizes to the cell cortex adjacent to the spindle poles and orients the mitotic spindle. In tubules, cell division may be oriented relative to two axes: longitudinal and apical-basal. Unexpectedly, in vivo examination of early-stage developing nephron tubules reveals that cell division is not oriented in the longitudinal (or planar-polarized) axis. However, cell division is oriented perpendicular to the apical-basal axis. Absence of afadin in vivo leads to misorientation of apical-basal cell division in nephron tubules. Together, these results support a model whereby afadin determines lumen placement by directing apical-basal spindle orientation, resulting in a continuous lumen and normal tubule morphogenesis.