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.

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.

SGLT2 inhibitors are the new mainstay of diabetes mellitus and cardiovascular diseases treatment. 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 sodium channels/transporters along the nephron in Dahl Salt-Sensitive (SS) rats, a model of salt-induced hypertension. Administration of dapagliflozin (Dapa) at 2 mg/kg/day via drinking water for 3 weeks 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-/creatinine ratios increased in Dapa-treated compared to vehicle-treated rats. To define the contribution of RAAS, we measured various hormones. Despite apparent effects on Na+ and Cl-/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 blotting, and patch-clamp. Neither mRNA nor protein expression levels of tested renal transporters (SGLT2, NHE3, NKCC2, NCC, and α-, β-, γ- ENaC subunits) changed significantly between groups. Furthermore, electrophysiological studies did not reveal any difference in Dapa treatment on the conductance and activity of ENaC. Our data suggest that SGLT2 inhibition in a non-diabetic model of salt-sensitive hypertension blunts the development of salt-induced hypertension by causing glucosuria and natriuresis without changes in RAAS and expression or activity of studied Na+ channels and transporters.

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.

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.

The effect of pharmacological blockade of ASIC1a channels on emotionally conditioned learning in rats with lithiumpilocarpine model of temporal lobe epilepsy was studied. The development of status epilepticus is known to impair both contextual and stimulated emotional learning. In our experiments, the painful effect was directly accompanied by freezing in animals of all groups, however, under conditions of induced epilepsy, its duration was shorter, and blocking ASIC1a channels neutralized this difference. The induction of epilepsy disrupted both contextual and stimulated emotional learning. Suppression of ASIC1a channels partially restored this process in animals with induced epilepsy. This means that pharmacological blocking of ASIC1a channels can have a positive therapeutic effect in correcting the emotional disorders associated with temporal lobe epilepsy.

Background and Purpose: Inwardly rectifying K + (K ir ) 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 K ir 4.1/K ir 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 K ir inhibitor, VU0134992, on the activity of K ir 4.1, K ir 4.1/K ir 5.1 and ENaC were tested using electrophysiological approaches in CHO cells transfected with respective channel subunits, cultured polarized epithelial mCCD cl1 cells and freshly isolated rat and human CCD tubules. To test the effect of pharmacological K ir 4.1/K ir 5.1 inhibition on electrolyte homeostasis in vivo and corresponding changes in distal tubule transport, Dahl salt-sensitive rats were injected with amitriptyline (15 mgÁkg À1 Áday À1 ) for 3 days.

No abstract