Positioned at the head of the nephron, the renal corpuscle generates a plasma ultrafiltrate to initiate urine formation. Three major cell types within the renal corpuscle, the glomerular mesangial cells, podocytes, and glomerular capillary endothelial cells communicate via endocrine and paracrine signaling mechanisms to maintain structure and function of the glomerular capillary network and filtration barrier. Ca2+ signaling mediated by several distinct plasma membrane Ca2+ channels modulates the functions of all three cell types. The last two decades have witnessed pivotal advances in understanding of Ca2+ channel function and regulation in glomerular cells, particularly non-voltage gated Ca2+ channels, in health and renal disease. This review summarizes the current knowledge of the physiological and pathological impact of non-voltage gated Ca2+ channel signaling in glomerular capillary endothelium, mesangial cells and podocytes. The main focus is on transient receptor potential and store-operated Ca2+ channels, but ionotropic N-methyl-D-aspartate receptors and purinergic 2X receptors also are discussed. This update of Ca2+ channel functions in the renal corpuscle and their cellular signaling cascades is intended to inform development of therapeutic strategies targeting these channels to treat kidney diseases, particularly diabetic nephropathy.

AbstractDiabetic kidney disease (DKD) is a devastating kidney disease and lacks effective therapeutic interventions. The present study was aimed to determine whether reconstituted high‐density lipoprotein (rHDL) ameliorated renal injury in eNOS−/− dbdb mice, a mouse model of DKD. Three groups of mice, wild type C57BLKS/J (non‐diabetes), eNOS−/− dbdb (diabetes), and eNOS−/− dbdb treated with rHDL (diabetes+rHDL) with both males and females were used. The rHDL nanoparticles were administered to eNOS−/− dbdb mice at Week 16 at 5 μg/g body weight in ~100 μL of saline solution twice per week for 4 weeks via retroorbital injection. We found that rHDL treatment significantly blunted progression of albuminuria and GFR decline observed in DKD mice. Histological examinations showed that the rHDLs significantly alleviated glomerular injury and renal fibrosis, and inhibited podocyte loss. Western blots and immunohistochemical examinations showed that increased protein abundances of fibronectin and collagen IV in the renal cortex of eNOS−/− dbdb mice were significantly reduced by the rHDLs. Taken together, the present study suggests a renoprotective effect of rHDLs on DKD.

Key PointsI-mfa is a multifunctional cytosolic protein and its function in kidney is unknown.The major finding in the present study was that I-mfa promoted glomerular filtration rate in both male and female mice.I-mfa suppressed contractile function of both human and mouse glomerular mesangial cells by decreasing TRPC1 channel protein abundance.BackgroundInhibitor of MyoD family A (I-mfa) is a cytosolic protein. Its function in the kidney is unknown. The aim of this study was to examine the regulatory role of I-mfa on GFR.MethodsGFR was measured by transdermal measurement of fluorescein isothiocyanate-sinitrin clearance in conscious wild-type (WT) and I-mfa knockout (KO) mice. Cell contractility was assessed in a single human or mouse mesangial cell. Single-cell RNA sequence, Western blot, and Ca2+ imaging were used to evaluate the effects of I-mfa on transient receptor potential canonical (TRPCs) at messenger, protein, and functional levels in mesangial cells.ResultsIn KO mice, GFR was significantly lower than that in WT mice. In WT mice, knocking down I-mfa selectively in mesangial cells using targeted nanoparticle/small interfering RNA delivery system significantly decreased GFR. In human mesangial cells, overexpression of I-mfa significantly blunted the angiotensin II (Ang II)-stimulated contraction, and knockdown of I-mfa significantly enhanced the contractile response. Consistently, the Ang II-induced contraction was significantly augmented in primary mesangial cells isolated from KO mice. The exaggerated response was restored by reintroducing I-mfa. Furthermore, single-cell RNA sequence showed an increase in trpc1 messenger, and Western blot showed an increase in TRPC1 protein abundance in I-mfa KO mouse mesangial cells. TRPC1 protein abundance was decreased in human embryonic kidney cells overexpressing I-mfa. Ca2+ imaging experiments showed that downregulation of I-mfa significantly enhanced Ang II-stimulated Ca2+ entry in human mesangial cells. Finally, TRPC1 inhibitor Pico145 significantly blunted Ang II-induced mesangial cell contraction.ConclusionsI-mfa positively regulated GFR by decreasing mesangial cell contractile function through inhibition of TRPC1-mediated Ca2+ signaling.

This study tested the hypothesis that overwhelming store-operated Ca 2+ entry is a novel mechanism contributing to high glucose- and angiotensin II-induced podocyte apoptosis and mitochondrial injury.

Purpose and hypothesis: Podocyte injury induced by Angiotensin II (Ang II) is the key factor contributing to proteinuria and glomerulopathy in many kidney diseases including diabetic nephropathy and hypertensive kidney disease. Recent studies suggest that mitochondria dysfunction mediates Ang II-induced podocyte injury. However, the mechanism for Ang II-induced podocyte mitochondria damage is poorly understood. Store-operated Ca2+ entry (SOCE) has multiple functions in both excitable and non-excitable cells. Ang II can activate SOCE by releasing endoplasmic reticulum Ca2+ through generation of IP3. In addition, Ang II also activates Transient Receptor Potential Conventional Member 6 (TRPC6) channel through diacylglycerol (DAG). Our previous study demonstrated that enhanced SOCE mediated high glucose-induced podocyte cytoskeleton remodeling. However, the role of SOCE in podocyte injury by Ang II is not clear. The present study was carried out to test the hypothesis that enhanced SOCE contributed to Ang II-induced podocyte apoptosis and mitochondria respiration dysfunction. Methods: All experiments were carried out using cultured immortalized human podocytes. BTP2 (4 μM) was used to inhibit SOCE. SAR 7334 (100 nM) was used to block TRPC6 channel. Podocyte apoptosis was determined by flow cytometry using Annexin V/Propidium iodide (PI) staining. The conventional whole-cell patch-clamp was performed to measure store-operated Ca2+ channel (SOC) currents. Mitochondrial respiration function and mitochondria ATP production were evaluated by oxygen consumption rate (OCR) using seahorse analysis. Results: Ang II (1 μM) evoked inward currents, which were significantly blunted by BTP2 (10 μM), an SOC channel blocker. Ang II (1 μM) treatments for 24 hours significantly increased podocyte apoptosis and reduced podocyte basal OCR, maximal OCR, spare capacity and mitochondria ATP production. All these responses were significantly blunted by BTP2. However, Ang II treatment for 30 min did not induce a significant response of OCR, but significantly reduced maximal respiration and spare capacity. These responses were also significantly inhibited by BTP2 (10 μM), but not by SAR 7334 (100 nM). Conclusion: SOCE contributed to Ang II-induced podocyte apoptosis and mitochondria respiratory dysfunction. Translational Project Award from American Heart Association (20TPA35500045, to RM); National Institute of Diabetes and Digestive and Kidney Disease (DK115424-01, to RM); Predoctoral Fellowship from American Heart Association (903925, to YT). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The present study was to examine sex and strain differences in glomerular filtration rate (GFR) and renal blood flow (RBF) in C57BL6, 129/Sv, and C57BLKS/J mice, three commonly used mouse strains in renal research. GFR was measured by transdermal measurement of FITC-sinitrin clearance in conscious mice. RBF was measured by a flow probe placed in the renal artery under an anesthetic state. In C57BL6 mice, there were no sex differences in both GFR and RBF. In 129/Sv mice, females had significantly greater GFR than males at age of 24 weeks, but not at 8 weeks. However, males had higher RBF and lower renal vascular resistance (RVR). Similar to 129/Sv, female C57BLKS/J had significantly greater GFR at both 8 and 24 weeks, lower RBF, and higher RVR than males. Across strains, male 129/Sv had lower GFR and higher RBF than male C57BL6, but no significant difference in GFR and greater RBF than male C57BLKS/J. No significant difference in GFR or RBF was observed between C57BL6 and C57BLKS/J mice. Deletion of eNOS in C57BLKS/J mice reduced GFR in both sexes, but decreased RBF in males. Furthermore, there were no sex differences in the severity of renal injury in eNOS-/- dbdb mice. Taken together, our study suggests that sex differences in renal hemodynamics in mice are strain and age dependent. eNOS was not involved in the sex differences in GFR, but in RBF. Furthermore, the sexual dimorphism did not impact the severity of renal injury in diabetic nephropathy.

An essential step in renal function entails the formation of an ultrafiltrate that is delivered to the renal tubules for subsequent processing. This process, known as glomerular filtration, is controlled by intrinsic regulatory systems and by paracrine, neuronal, and endocrine signals that converge onto glomerular cells. In addition, the characteristics of glomerular fluid flow, such as the glomerular filtration rate and the glomerular filtration fraction, play an important role in determining blood flow to the rest of the kidney. Consequently, disease processes that initially affect glomeruli are the most likely to lead to end-stage kidney failure. The cells that comprise the glomerular filter, especially podocytes and mesangial cells, express many different types of ion channels that regulate intrinsic aspects of cell function and cellular responses to the local environment, such as changes in glomerular capillary pressure. Dysregulation of glomerular ion channels, such as changes in TRPC6, can lead to devastating glomerular diseases, and a number of channels, including TRPC6, TRPC5, and various ionotropic receptors, are promising targets for drug development. This review discusses glomerular structure and glomerular disease processes. It also describes the types of plasma membrane ion channels that have been identified in glomerular cells, the physiological and pathophysiological contexts in which they operate, and the pathways by which they are regulated and dysregulated. The contributions of these channels to glomerular disease processes, such as focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy, as well as the development of drugs that target these channels are also discussed.

Studies over the last decade have markedly broadened our understanding of store-operated Ca2+ channels (SOCs) and their roles in kidney diseases and podocyte dysfunction. Podocytes are terminally differentiated glomerular visceral epithelial cells which are tightly attached to the glomerular capillary basement membrane. Podocytes and their unique foot processes (pedicels) constitute the outer layer of the glomerular filtration membrane and the final barrier preventing filtration of albumin and other plasma proteins. Diabetic nephropathy and other renal diseases are associated with podocyte injury and proteinuria. Recent evidence demonstrates a pivotal role of store-operated Ca2+ entry (SOCE) in maintaining structural and functional integrity of podocytes. This article reviews the current knowledge of SOCE and its contributions to podocyte physiology. Recent studies of the contributions of SOC dysfunction to podocyte injury in both cell culture and animal models are discussed, including work in our laboratory. Several downstream signaling pathways mediating SOC function in podocytes also are examined. Understanding the pivotal roles of SOC in podocyte health and disease is essential, as SOCE-activated signaling pathways are potential treatment targets for podocyte injury-related kidney diseases.

The primary focus of this review is lipoprotein-based drug carriers, more specifically, high-density lipoprotein (HDL) type nanoparticles (NPs). These nanostructures are discussed regarding their suitability for clinical applications, particularly for cancer therapy. Poor solubility and insufficient capability to selectively target malignant tumors represent significant challenges facing many anticancer drugs. Nevertheless, we and others have found that most, if not all, of these difficulties, can be overcome by incorporating drugs into lipoprotein nanocarriers1. While not a novel approach, as HDL type NPs have been documented to deliver anticancer agents to cancer cells effectively and tumors2,3,4,5, including those that, on their own (without facilitation), exhibited less than desirable therapeutic efficacy6, due to their desirable features (see below), HDL type drug carriers, at least in our view, hold tremendous promise as facilitators of cancer chemotherapy. One of the key aspects of the HDL-type NP-facilitated drug transport is the receptor-mediated uptake of the payload from the NPs7,8. Consequently, in this review, major emphasis is placed on monitoring the expression of the scavenger receptor type B1 (SR-B1) as a potentially valuable tool for the pre-treatment selection of patients regarding their suitability for advanced, personalized chemotherapy. The main emphasis in this article is on developing novel cancer therapeutics, while approaches for treating other diseases via lipoprotein nanocarriers are briefly discussed.

These data indicate that increased expression of renal TLR7 and TNF-α is associated with renal injury and hypertension in female SLE mice. In addition, these data suggest a potential sex difference in the pathogenesis of SLE in males. Therapeutic strategies targeting the TLR7 molecular pathway should be further investigated in both female and male lupus nephritis.

Podocyte injury induced by hyperglycemia is the main cause of kidney dysfunction in diabetic nephropathy. However, the underlying mechanism is unclear. Store-operated Ca2+ entry (SOCE) regulates a diversity of cellular processes in a variety of cell types. Calpain, a Ca2+-dependent cysteine protease, was recently shown to be involved in podocyte injury. In the present study, we sought to determine whether increased SOCE contributed to high glucose (HG)-induced podocyte injury through activation of the calpain pathway. In cultured human podocytes, whole-cell patch clamp indicated the presence of functional store-operated Ca2+ channels, which are composed of Orai1 proteins and mediate SOCE. Western blots showed that HG treatment increased the protein abundance of Orai1 in a dose-dependent manner. Consistently, calcium imaging experiments revealed that SOCE was significantly enhanced in podocytes following HG treatment. Furthermore, HG treatment caused overt podocyte F-actin disorganization as well as a significant decrease in nephrin protein abundance, both of which are indications of podocyte injury. These podocyte injury responses were significantly blunted by both pharmacological inhibition of Orai1 using the small molecule inhibitor BTP2 or by genetic deletion of Orai1 using CRISPR-Cas9 lentivirus. Moreover, activation of SOCE by thapsigargin, an inhibitor of Ca2+ pump on the endoplasmic/sarcoplasmic reticulum membrane, significantly increased the activity of calpain, which was inhibited by BTP2. Finally, the calpain-1/calpain-2 inhibitor calpeptin significantly blunted the nephrin protein reduction induced by HG treatment. Taken together, our results suggest that enhanced signaling via an Orai1/SOCE/Calpain axis contributes to HG-induced podocyte injury.

Hypertension is prevalent in patients with systemic lupus erythematosus (SLE). The goal of the current study is to track the pathogenesis of hypertension and renal injury in SLE, identify contributory mechanisms, and highlight differences in disease development among sexes. Mean arterial pressure was measured in conscious male and female SLE (NZBWF1) and control (NZW) mice at 34-35 wk of age using indwelling arterial catheters. Measures of renal injury, renal inflammation, and renal hemodynamics were used to monitor the potential contributors to latent sex differences. Both male and female SLE mice were hypertensive at 35 wk of age, and the hypertension was linked to renal injury in females, but not in males. A known contributor of renal pathology in SLE, Toll-like receptor (TLR)-7, and its downstream effector, the proinflammatory cytokine tumor necrosis factor (TNF)-α, were lower in male SLE mice than in females. Male SLE mice also had higher glomerular filtration rate (GFR) and lower renal vascular resistance (RVR) than females. Our data suggest that although hypertension in female SLE mice is associated with renal mechanisms, hypertension in male SLE mice may develop independent of renal changes. Future studies will continue to dissect sex-specific factors that should be considered when treating patients with hypertension with underlying chronic inflammation and/or autoimmunity.NEW & NOTEWORTHY There is a high prevalence of hypertension in male and female SLE; however, male SLE mice are hypertensive without renal involvement. The development of hypertension in female SLE mice is renocentric and strongly associated with injurious renal mechanisms like the TLR-7→TNF-α pathway. This clear difference in the pathogenesis among the sexes could have a significant impact on how we treat patients with hypertension with underlying chronic autoimmune/inflammatory diseases.

Neogenin, a transmembrane receptor, was recently found in kidney cells and immune cells. However, the function of neogenin signaling in kidney is not clear. Mesangial cells (MCs) are a major source of extracellular matrix (ECM) proteins in glomerulus. In many kidney diseases, MCs are impaired and manifest myofibroblast phenotype. Overproduction of ECM by the injured MCs promotes renal injury and accelerates the progression of kidney diseases. The present study aimed to determine if neogenin receptor was expressed in MCs and if the receptor signaling regulated ECM protein production by MCs. We showed that neogenin was expressed in the glomerular MCs. Deletion of neogenin using CRISPR/Cas9 lentivirus system significantly reduced the abundance of fibronectin, an ECM protein. Netrin-1, a ligand for neogenin, also significantly decreased fibronectin production by MCs and decreased neogenin protein expression in MCs. Furthermore, treatment of human MCs with high glucose (HG, 25 mM) significantly increased the protein abundance of neogenin as early as 8 h. Consistently, neogenin expression in glomerulus significantly increased in the eNOS-/-db/db diabetic mice starting as early as the age of 8 wk and this increase sustained at least to the age of 24 wk. We further found that the HG-induced increase in neogenin abundance was blunted by antioxidant PEG-catalase and N-acetyl cysteine. Taken together, our results suggest a new mechanism of regulation of fibronectin production by MCs. This previously unrecognized neogenin-fibronectin pathway may contribute to glomerular injury responses during the course of diabetic nephropathy.

Enhanced SOCE contributed to HG induced podocyte injury and mitochondrial dysfunction.

Purpose Diabetic Nephropathy is one of the major complications of diabetes mellitus. Hyperglycemia is a known initiator of diabetes mellitus. Accumulating evidence suggests that podocyte injury is tightly associated with the onset and progression of diabetic nephropathy. However, the mechanisms underlying podocyte injury induced by hyperglycemia or high glucose (HG) is poorly understood. Store-operated calcium entry (SOCE) is a multifunctional signaling pathway in many cell types. However, its role in podocyte injury in the settings of diabetes is not known. The aim of the present study was to examine if SOCE mediated HG-induced podocyte injury by impairing mitochondrial function. Methods All experiments were carried out in cultured immortalized human podocytes. Western blot was conducted to evaluate protein abundance of Orai1 (the channel protein mediating SOCE) and nephrin (a podocyte specific protein). Calcium imaging was used to analyze SOCE. TMRE fluorescence was used to probe the mitochondria membrane potential (MMP). Results HG (25mM) significantly increased podocyte Orai1 protein abundance for time periods ranging from 2 to 12 hours. This HG effect was dose dependent. Consistently, Ca2+ imaging experiments showed that HG treatment (25 mM for 12 hours) significantly enhanced podocyte SOCE. Furthermore, the abundance of nephrin protein decreased in podocytes after exposure to HG, indicating podocyte injury in the presence of ambient HG. However, the HG-induced decrease of nephrin was blunted by BTP2 (an SOCE inhibitor, 4 µM). Moreover, HG treatment (25 mM for 24 hours) decreased MMP, indicating damage of mitochondria by HG. The decrease of MMP was prevented by BTP2, suggesting the contribution of SOCE to the detrimental effect of HG treatment. Conclusion The present study suggests that upregulated SOCE contributes to HG-induced podocyte injury, possibly by impairing mitochondrial function.

Research conducted over the last two decades has dramatically advanced the understanding of store-operated calcium channels (SOCC) and their impact on renal function. Kidneys contain many types of cells, including those specialized for glomerular filtration (fenestrated capillary endothelium, podocytes), water and solute transport (tubular epithelium), and regulation of glomerular filtration and renal blood flow (vascular smooth muscle cells, mesangial cells). The highly integrated function of these myriad cells effects renal control of blood pressure, extracellular fluid volume and osmolality, electrolyte balance, and acid–base homeostasis. Many of these cells are regulated by Ca2+signaling. Recent evidence demonstrates that SOCCs are major Ca2+entry portals in several renal cell types. SOCC is activated by depletion of Ca2+stores in the sarco/endoplasmic reticulum, which communicates with plasma membrane SOCC via the Ca2+sensor Stromal Interaction Molecule 1 (STIM1). Orai1 is recognized as the main pore-forming subunit of SOCC in the plasma membrane. Orai proteins alone can form highly Ca2+selective SOCC channels. Also, members of the Transient Receptor Potential Canonical (TRPC) channel family are proposed to form heteromeric complexes with Orai1 subunits, forming SOCC with low Ca2+selectivity. Recently, Ca2+entry through SOCC, known as store-operated Ca2+entry (SOCE), was identified in glomerular mesangial cells, tubular epithelium, and renovascular smooth muscle cells. The physiological and pathological relevance and the characterization of SOCC complexes in those cells are still unclear. In this review, we summarize the current knowledge of SOCC and their roles in renal glomerular, tubular and vascular cells, including studies from our laboratory, emphasizing SOCE regulation of fibrotic protein deposition. Understanding the diverse roles of SOCE in different renal cell types is essential, as SOCC and its signaling pathways are emerging targets for treatment of SOCE-related diseases.