RAS-mediated nitric oxide signaling in podocytes.

Nitric oxide (NO) is widely recognized for its role in regulating renal function and blood pressure. However, the precise mechanisms by which NO affects renal epithelial cells remain understudied. Our previous research has shown that NO signaling in glomerular podocytes can be initiated by Angiotensin II (Ang II) but not by ATP. This study aims to elucidate the crucial interplay between the renin-angiotensin system (RAS) and NO production in podocytes. To conduct our research, we utilized cultured human podocytes and freshly isolated rat glomeruli. A variety of RAS peptides were employed, alongside confocal microscopy, to detect NO production and NO/Ca2+ crosstalk. Dynamic changes in the podocyte cytoskeleton, mediated by RAS-NO intracellular signaling, were observed using fluorescent labeling for F-actin and scanning probe microscopy. The experiments demonstrated that Ang II and Ang III generated high levels of NO through the activation of the Angiotensin II Type 2 Receptor (AT2R). We did not detect functional MAS receptor presence in podocytes, and the moderate NO response to Ang 1-7 was also mediated through AT2R. Furthermore, NO production impacted intracellular Ca2+ signaling and correlated with an increase in podocyte volume and growth. Scanning probe experiments revealed that AT2R activation and the corresponding NO generation are responsible for the protrusion of podocyte lamellipodia. Taken together, our data indicate that AT2R activation enhances NO production in podocytes and subsequently mediates changes in Ca2+ signaling and podocyte volume dynamics. These mechanisms may play a significant role in both physiological and pathophysiological interactions between the RAS and podocytes.

ß-Arrestin pathway activation by selective ATR1 agonism promotes calcium influx in podocytes, leading to glomerular damage.

Angiotensin receptor blockers (ARBs) are the first-line treatment for hypertension; they act by inhibiting signaling through the angiotensin 1 receptor (AT1R). Recently, a novel biased AT1R agonist, TRV120027 (TRV), which selectively activates the ß-arrestin cascade and blocks the G-protein-coupled receptor pathway has been proposed as a potential blood pressure medication. Here, we explored the effects of TRV and associated ß-arrestin signaling in podocytes, essential cells of the kidney filter. We used human podocyte cell lines to determine ß-arrestin's involvement in calcium signaling and cytoskeletal reorganization and Dahl SS rats to investigate the chronic effects of TRV administration on glomerular health. Our experiments indicate that the TRV-activated ß-arrestin pathway promotes the rapid elevation of intracellular Ca2+ in a dose-dependent manner. Interestingly, the amplitude of ß-arrestin-mediated Ca2+ influx was significantly higher than the response to similar Ang II concentrations. Single-channel analyses show rapid activation of transient receptor potential canonical (TRPC) channels following acute TRV application. Furthermore, the pharmacological blockade of TRPC6 significantly attenuated the ß-arrestin-mediated Ca2+ influx. Additionally, prolonged activation of the ß-arrestin pathway in podocytes resulted in pathological actin cytoskeleton rearrangements, higher apoptotic cell markers, and augmented glomerular damage. TRV-activated ß-arrestin signaling in podocytes may promote TRPC6 channel-mediated Ca2+ influx, foot process effacement, and apoptosis, possibly leading to severe defects in glomerular filtration barrier integrity and kidney health. Under these circumstances, the potential therapeutic application of TRV for hypertension treatment requires further investigation to assess the balance of the benefits versus possible deleterious effects and off-target damage.

Metabolic Markers and Association of Biological Sex in Lupus Nephritis.

Lupus nephritis (LN) is a serious complication for many patients who develop systemic lupus erythematosus, which primarily afflicts women. Our studies to identify biomarkers and the pathogenic mechanisms underlying LN will provide a better understanding of disease progression and sex bias, and lead to identification of additional potential therapeutic targets. The glycosphingolipid lactosylceramide (LacCer) and N-linked glycosylated proteins (N-glycans) were measured in urine and serum collected from LN and healthy control (HC) subjects (10 females and 10 males in each group). The sera from the LN and HC subjects were used to stimulate cytokine secretion and intracellular Ca2+ flux in female- and male-derived primary human renal mesangial cells (hRMCs). Significant differences were observed in the urine of LN patients compared to HCs. All major LacCers species were significantly elevated and differences between LN and HC were more pronounced in males. 72 individual N-glycans were altered in LN compared to HC and three N-glycans were significantly different between the sexes. In hRMCs, Ca2+ flux, but not cytokine secretion, was higher in response to LN sera compared to HC sera. Ca2+ flux, cytokine secretion, and glycosphingolipid levels were significantly higher in female-derived compared to male-derived hRMCs. Relative abundance of some LacCers and hexosylceramides were higher in female-derived compared to male-derived hRMCs. Urine LacCers and N-glycome could serve as definitive LN biomarkers and likely reflect renal disease activity. Despite higher sensitivity of female hRMCs, males may experience greater increases in LacCers, which may underscore worse disease in males. Elevated glycosphingolipid metabolism may poise renal cells to be more sensitive to external stimuli.

Scanning ion conductance microscopy of live human glomerulus.

Podocyte damage is a hallmark of glomerular diseases, such as focal segmental glomerulosclerosis, typically associated with marked albuminuria and progression of renal pathology. Podocyte structural abnormalities and loss are also linked to minimal change disease and more common diabetic kidney disease. Here we applied the first-time scanning ion conductance microscopy (SICM) technique to assess the freshly isolated human glomerulus's topology. SICM provides a unique opportunity to evaluate glomerulus podocytes as well as other nephron structural segments with electron microscopy resolution but in live samples. Shown here is the application of the SICM method in the live human glomerulus, which provides proof of principle for future dynamic analysis of membrane morphology and various functional parameters in living cells.

Vibrodissociation method for isolation of defined nephron segments from human and rodent kidneys.

Our current knowledge of the properties of renal ion channels responsible for electrolytes and cell energy homeostasis mainly relies on rodent studies. However, it has not been established yet to what extent their characteristics can be generalized to those of humans. The present study was designed to develop a standardized protocol for the isolation of well-preserved glomeruli and renal tubules from rodent and human kidneys and to assess the functional suitability of the obtained materials for physiological studies. Separation of nephron segments from human and rodent kidneys was achieved using a novel vibrodissociation technique. The integrity of isolated renal tubules and glomeruli was probed via electrophysiological analysis and fluorescence microscopy, and the purity of the collected fractions was confirmed using quantitative RT-PCR with gene markers for specific cell types. The developed approach allows rapid isolation of well-preserved renal tubules and glomeruli from human and rodent kidneys amenable for electrophysiological, Ca2+ imaging, and omics studies. Analysis of the basic electrophysiological parameters of major K+ and Na+ channels expressed in human cortical collecting ducts revealed that they exhibited similar biophysical properties as previously reported in rodent studies. Using vibrodissociation for nephron segment isolation has several advantages over existing techniques: it is less labor intensive, requires little to no enzymatic treatment, and produces large quantities of well-preserved experimental material in pure fractions. Applying this method for the separation of nephron segments from human and rodent kidneys may be a powerful tool for the indepth assessment of kidney function in health and disease.

Progression of diabetic kidney disease in T2DN rats.

Diabetic kidney disease (DKD) is one of the leading pathological causes of decreased renal function and progression to end-stage kidney failure. To explore and characterize age-related changes in DKD and associated glomerular damage, we used a rat model of type 2 diabetic nephropathy (T2DN) at 12 wk and older than 48 wk. We compared their disease progression with control nondiabetic Wistar and diabetic Goto-Kakizaki (GK) rats. During the early stages of DKD, T2DN and GK animals revealed significant increases in blood glucose and kidney-to-body weight ratio. Both diabetic groups had significantly altered renin-angiotensin-aldosterone system function. Thereafter, during the later stages of disease progression, T2DN rats demonstrated a remarkable increase in renal damage compared with GK and Wistar rats, as indicated by renal hypertrophy, polyuria accompanied by a decrease in urine osmolarity, high cholesterol, a significant prevalence of medullary protein casts, and severe forms of glomerular injury. Urinary nephrin shedding indicated loss of the glomerular slit diaphragm, which also correlates with the dramatic elevation in albuminuria and loss of podocin staining in aged T2DN rats. Furthermore, we used scanning ion microscopy topographical analyses to detect and quantify the pathological remodeling in podocyte foot projections of isolated glomeruli from T2DN animals. In summary, T2DN rats developed renal and physiological abnormalities similar to clinical observations in human patients with DKD, including progressive glomerular damage and a significant decrease in renin-angiotensin-aldosterone system plasma levels, indicating these rats are an excellent model for studying the progression of renal damage in type 2 DKD.

PAR1-mediated Non-periodical Synchronized Calcium Oscillations in human Mesangial Cells.

Mesangial cells offer structural support to the glomerular tuft and regulate glomerular capillary flow through their contractile capabilities. These cells undergo phenotypic changes, such as proliferation and mesangial expansion, resulting in abnormal glomerular tuft formation and reduced capillary loops. Such adaptation to the changing environment is commonly associated with various glomerular diseases, including diabetic nephropathy and glomerulonephritis. Thrombin-induced mesangial remodeling was found in diabetic patients, and expression of the corresponding protease-activated receptors (PARs) in the renal mesangium was reported. However, the functional PAR-mediated signaling in mesangial cells was not examined. This study investigated protease-activated mechanisms regulating mesangial cell calcium waves that may play an essential role in the mesangial proliferation or constriction of the arteriolar cells. Our results indicate that coagulation proteases like thrombin induce synchronized oscillations in cytoplasmic Ca2+ concentration of mesangial cells. The oscillations required PAR1 GPCRs-related activation, but not a PAR4, and were further mediated presumably through store-operated calcium entry and TRPC3 channel activity. Understanding thrombin signaling pathways and their relation to mesangial cells' contractile or synthetic (proliferative) phenotype may play a role in the development of chronic kidney disease and requires further investigation.

Crosstalk between epithelial sodium channels (ENaC) and basolateral potassium channels (Kir 4.1/Kir 5.1) in the cortical collecting duct.

BACKGROUND AND PURPOSE: Inwardly rectifying K+ (Kir ) channels located on the basolateral membrane of epithelial cells of the distal nephron play a crucial role in K+ handling and BP control, making these channels an attractive target for the treatment of hypertension. The purpose of the present study was to determine how the inhibition of basolateral Kir 4.1/Kir 5.1 heteromeric K+ channel affects epithelial sodium channel (ENaC)-mediated Na+ transport in the principal cells of cortical collecting duct (CCD). EXPERIMENTAL APPROACH: The effect of fluoxetine, amitriptyline and recently developed Kir inhibitor, VU0134992, on the activity of Kir 4.1, Kir 4.1/Kir 5.1 and ENaC were tested using electrophysiological approaches in CHO cells transfected with respective channel subunits, cultured polarized epithelial mCCDcl1 cells and freshly isolated rat and human CCD tubules. To test the effect of pharmacological Kir 4.1/Kir 5.1 inhibition on electrolyte homeostasis in vivo and corresponding changes in distal tubule transport, Dahl salt-sensitive rats were injected with amitriptyline (15 mg·kg-1 ·day-1 ) for 3 days. KEY RESULTS: We found that inhibition of Kir 4.1/Kir 5.1, but not the Kir 4.1 channel, depolarizes the cell membrane, induces the elevation of intracellular Ca2+ concentration and suppresses ENaC activity. Furthermore, we demonstrate that amitriptyline administration leads to a significant drop in plasma K+ level, triggering sodium excretion and diuresis. CONCLUSION AND IMPLICATIONS: The present data uncover a specific role of the Kir 4.1/Kir 5.1 channel in the modulation of ENaC activity and emphasize the potential for using Kir 4.1/Kir 5.1 inhibitors to regulate electrolyte homeostasis and BP.

Inhibition of protease-activated receptor 1 ameliorates behavioral deficits and restores hippocampal synaptic plasticity in a rat model of status epilepticus.

The blood-brain barrier (BBB) is a unique structure that controls substances exchange between the systemic circulation and the brain. Disruption of its integrity contributes to the development and progression of a variety of brain disorders including stroke, epilepsy and neurodegenerative diseases. It was shown that intracerebral thrombin level substantially increases following status epilepticus (SE). Inhibition of protease-activated receptor 1 (PAR1), the major thrombin receptor in the brain, produces an anti-epileptogenic and neuroprotective effects in an experimental model of temporal lobe epilepsy (TLE). Since serine proteases and PAR1 are implicated in the synaptic plasticity and memory formation, the aim of the present study was to elucidate the involvement of PAR1 in synaptic plasticity and behavior deficits following SE. Using lithium-pilocarpine model of TLE, we demonstrate that inhibition of PAR1 rescues SE-induced synaptic plasticity deficits in CA1 region of hippocampus. Although treatment with PAR1 antagonist does not ameliorate spatial learning deficits, it attenuates anxiolytic-like behavior in experimental rats after SE. Taken together; our data suggest an important role of PAR1 in SE-induced synaptic and behavioral alterations and provide a new insight into cellular mechanisms underlying behavioral impairments associated with epilepsy.