Protease-Activated Receptor 1-Mediated Damage of Podocytes in Diabetic Nephropathy.

There is clinical evidence that increased urinary serine proteases are associated with the disease severity in the setting of diabetic nephropathy (DN). Elevation of serine proteases may mediate [Ca2+]i dynamics in podocytes through the protease-activated receptors (PARs) pathway, including associated activation of nonspecific cation channels. Cultured human podocytes and freshly isolated glomeruli were used for fluorescence and immunohistochemistry stainings, calcium imaging, Western blot analysis, scanning ion conductance microscopy, and patch clamp analysis. Goto-Kakizaki, Wistar, type 2 DN (T2DN), and a novel PAR1 knockout on T2DN rat background rats were used to test the importance of PAR1-mediated signaling in DN settings. We found that PAR1 activation increases [Ca2+]i via TRPC6 channels. Both human cultured podocytes exposed to high glucose and podocytes from freshly isolated glomeruli of T2DN rats had increased PAR1-mediated [Ca2+]i compared with controls. Imaging experiments revealed that PAR1 activation plays a role in podocyte morphological changes. T2DN rats exhibited a significantly higher response to thrombin and urokinase. Moreover, the plasma concentration of thrombin in T2DN rats was significantly elevated compared with Wistar rats. T2DNPar1-/- rats were embryonically lethal. T2DNPar1+/- rats had a significant decrease in glomerular damage associated with DN lesions. Overall, these data provide evidence that, during the development of DN, elevated levels of serine proteases promote an excessive [Ca2+]i influx in podocytes through PAR1-TRPC6 signaling, ultimately leading to podocyte apoptosis, the development of albuminuria, and glomeruli damage. ARTICLE HIGHLIGHTS: Increased urinary serine proteases are associated with diabetic nephropathy. During the development of diabetic nephropathy in type 2 diabetes, the elevation of serine proteases could overstimulate protease-activated receptor 1 (PAR1). PAR1 signaling is involved in the development of DN via TRPC6-mediated intracellular calcium signaling. This study provides fundamental knowledge that can be used to develop efficient therapeutic approaches targeting serine proteases or corresponding PAR pathways to prevent or slow the progression of diabetes-associated kidney diseases.

SGLT2 inhibition effect on salt-induced hypertension, RAAS, and Na+ transport in Dahl SS rats.

Na+-glucose cotransporter-2 (SGLT2) inhibitors are the new mainstay of treatment for diabetes mellitus and cardiovascular diseases. Despite the remarkable benefits, the molecular mechanisms mediating the effects of SGLT2 inhibitors on water and electrolyte balance are incompletely understood. The goal of this study was to determine whether SGLT2 inhibition alters blood pressure and kidney function via affecting the renin-angiotensin-aldosterone system (RAAS) and Na+ channels/transporters along the nephron in Dahl salt-sensitive rats, a model of salt-induced hypertension. Administration of dapagliflozin (Dapa) at 2 mg/kg/day via drinking water for 3 wk blunted the development of salt-induced hypertension as evidenced by lower blood pressure and a left shift of the pressure natriuresis curve. Urinary flow rate, glucose excretion, and Na+- and Cl--to-creatinine ratios increased in Dapa-treated compared with vehicle-treated rats. To define the contribution of the RAAS, we measured various hormones. Despite apparent effects on Na+- and Cl--to-creatinine ratios, Dapa treatment did not affect RAAS metabolites. Subsequently, we assessed the effects of Dapa on renal Na+ channels and transporters using RT-PCR, Western blot analysis, and patch clamp. Neither mRNA nor protein expression levels of renal transporters (SGLT2, Na+/H+ exchanger isoform 3, Na+-K+-2Cl- cotransporter 2, Na+-Cl- cotransporter, and α-, ß-, and γ-epithelial Na+ channel subunits) changed significantly between groups. Furthermore, electrophysiological experiments did not reveal any difference in Dapa treatment on the conductance and activity of epithelial Na+ channels. Our data suggest that SGLT2 inhibition in a nondiabetic model of salt-sensitive hypertension blunts the development of salt-induced hypertension by causing glucosuria and natriuresis without changes in the RAAS or the expression or activity of the main Na+ channels and transporters.NEW & NOTEWORTHY The present study indicates that Na+-glucose cotransporter-2 (SGLT2) inhibition in a nondiabetic model of salt-sensitive hypertension blunts the development and magnitude of salt-induced hypertension. Chronic inhibition of SGLT2 increases glucose and Na+ excretion without secondary effects on the expression and function of other Na+ transporters and channels along the nephron and hormone levels in the renin-angiotensin-aldosterone system. These data provide novel insights into the effects of SGLT2 inhibitors and their potential use in hypertension.

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.

Cytoskeleton Rearrangements Modulate TRPC6 Channel Activity in Podocytes.

The actin cytoskeleton of podocytes plays a central role in the functioning of the filtration barrier in the kidney. Calcium entry into podocytes via TRPC6 (Transient Receptor Potential Canonical 6) channels leads to actin cytoskeleton rearrangement, thereby affecting the filtration barrier. We hypothesized that there is feedback from the cytoskeleton that modulates the activity of TRPC6 channels. Experiments using scanning ion-conductance microscopy demonstrated a change in migration properties in podocyte cell cultures treated with cytochalasin D, a pharmacological agent that disrupts the actin cytoskeleton. Cell-attached patch-clamp experiments revealed that cytochalasin D increases the activity of TRPC6 channels in CHO (Chinese Hamster Ovary) cells overexpressing the channel and in podocytes from freshly isolated glomeruli. Furthermore, it was previously reported that mutation in ACTN4, which encodes α-actinin-4, causes focal segmental glomerulosclerosis and solidifies the actin network in podocytes. Therefore, we tested whether α-actinin-4 regulates the activity of TRPC6 channels. We found that co-expression of mutant α-actinin-4 K255E with TRPC6 in CHO cells decreases TRPC6 channel activity. Therefore, our data demonstrate a direct interaction between the structure of the actin cytoskeleton and TRPC6 activity.

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.

Role of opioid signaling in kidney damage during the development of salt-induced hypertension.

Opioid use is associated with predictors of poor cardiorenal outcomes. However, little is known about the direct impact of opioids on podocytes and renal function, especially in the context of hypertension and CKD. We hypothesize that stimulation of opioid receptors (ORs) contributes to dysregulation of intracellular calcium ([Ca2+]i) homeostasis in podocytes, thus aggravating the development of renal damage in hypertensive conditions. Herein, freshly isolated glomeruli from Dahl salt-sensitive (SS) rats and human kidneys, as well as immortalized human podocytes, were used to elucidate the contribution of specific ORs to calcium influx. Stimulation of κ-ORs, but not µ-ORs or δ-ORs, evoked a [Ca2+]i transient in podocytes, potentially through the activation of TRPC6 channels. κ-OR agonist BRL52537 was used to assess the long-term effect in SS rats fed a high-salt diet. Hypertensive rats chronically treated with BRL52537 exhibited [Ca2+]i overload in podocytes, nephrinuria, albuminuria, changes in electrolyte balance, and augmented blood pressure. These data demonstrate that the κ-OR/TRPC6 signaling directly influences podocyte calcium handling, provoking the development of kidney injury in the opioid-treated hypertensive cohort.

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.

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.