Injury to podocytes is considered a major contributor to diabetic kidney disease: their loss causes proteinuria and progressive glomerulosclerosis. Podocyte depletion may result from improper calcium handling due to abnormal activation of the calcium permeant TRPC (Transient Receptor Potential Canonical) channels. Angiotensin II (Ang II) levels are found to be elevated in diabetes; furthermore, it was reported that Ang II causes activation of TRPC6 in podocytes. We hypothesized here that Ang II-mediated calcium influx is aggravated in the podocytes under the conditions of type 1 diabetic nephropathy (DN). Diabetes was induced in the Dahl Salt-Sensitive rats by an injection of streptozotocin (STZ-SS). Eleven weeks post treatment was sufficient for the animals to develop hyperglycemia, excessive urination, weight loss, microalbuminuria, nephrinuria and display renal histological lesions typical for patients with DN. Patch-clamp electrophysiology performed on podocytes of the freshly isolated glomeruli showed enhanced basal TRPC channel activity in the STZ-SS rats, and increased response to Ang II; total calcium influx triggered by Ang II application was also augmented in podocytes of these rats. Our studies have a strong potential for advancing the understanding of TRPC-mediated effects on podocytopenia in DN initiation.

Injury to podocytes (terminally differentiated epithelial cells of the glomeruli) is considered a major contributor to diabetic kidney disease, when podocyte loss causes proteinuria and progressive glomerulosclerosis. Podocyte depletion may result from improper calcium handling in these cells, f.i. due to abnormal activation of the TRPC channels stimulated by Angiotensin II (Ang II). The goal of this study was to assess the ramifications of type 1 diabetes on Ang II mediated calcium influx in the podocytes. Diabetes was induced in the 6 week old Dahl salt‐sensitive rats by an injection of STZ, which resulted in weight loss, excessive urination and renal damage typical for diabetic nephropathy. During next 12 weeks rats were monitored to ensure hyperglycemia; biochemical analyses showed progressive proteinuria and high nephrin excretion, whereas excretion of Na+ and K+ remained normal. Patch‐clamp electrophysiology performed on podocytes of the isolated glomeruli showed increased basal TRPC6 channel activity (FNPo) in the STZ‐treated animals compared to controls (41% and 18% of patches, respectively), and a 5‐fold elevation of the channels open probability in response to 10 µM Ang II (the conductance (18 pS) and reversal potential of the channel (0 mV) remained unchanged). Ratiometric confocal measurements in glomeruli loaded with Fluo4/FuraRed dyes showed increased calcium influx in response to Ang II in diabetic rats compared to control animals. We can conclude from these data that Ang II‐dependent TRPC6 channels regulation plays a critical role in the glomerular filtration barrier destruction during type 1 diabetes.

Calcium flux in the podocytes is critical for their physiological function; excessive calcium in these cells can result in glomeruli damage, proteinuria and reduced GFR. P2 receptors are located throughout the kidney, and upon binding of ATP they activate calcium influx that triggers a plethora of intracellular processes. Our previous data have demonstrated that the major P2 receptor in the Sprague Dawley rat podocytes is P2Y1. Diabetic conditions have been shown to cause glomeruli injury and alter purinergic receptors profile in the kidney. The goal of the current study was to identify the P2 receptors responsible for calcium flux in the podocytes of the rats with type I diabetes. Type I diabetes was induced in the Dahl salt‐sensitive (SS) rats by an injection of streptozocin followed by an insulin implant, which results in the development of hyperfiltration, progressive proteinuria and renal damage typical for diabetic nephropathy. After 6 weeks of treatment, the glomeruli of the rats were isolated by differential sieving, loaded with Fluo‐4/FuraRed calcium dyes and analyzed with real‐time ratiometric fluorescent measurements. Pharmacological agonists and antagonists of the P2X and P2Y receptors were utilized to determine P2 receptors profile under diabetic conditions. This study will define the P2 receptors involved in the pathogenesis of the glomerular injury in diabetes, which could lead to a targeted treatment for renal complications associated with this disease.

Podocytes play a key role in the development of nephropathy: podocyte depletion results in reduced GFR and progressive glomerulosclerosis. Circulating factors can activate calcium influx in these cells triggering processes that lead to podocyte loss. This study was aimed at developing a proper model of diabetic nephropathy (DN) on the basis of the Dahl Salt‐Sensitive (SS) rat that could be further used for assessing calcium signaling in podocytes. Type I diabetes was induced in 6 week old SS rats by an injection of 75 mg/kg streptozotocin (STZ) which caused an elevation of blood glucose up to 700 mg/dL; this was then decreased to 350 mg/dL by an insulin pellet implant. Rats were monitored throughout the next 6 or 12 weeks; urine analyses revealed progressive proteinuria, whereas sodium and potassium fractional excretion levels were normal in both control and diabetic groups (0.3 and 14 for fractional excretion of Na and K, respectively). However, 6 weeks after the STZ injection was not sufficient to develop histological features characteristic in DN, whereas the 12 week treatment resulted in typical DN kidney damage. Urinary ELISA showed a 50‐fold increase in nephrin excretion in the 12 week hyperglycemic animals, which is indicative of podocyte depletion and foot processes effacement. At the end of the experiment rat glomeruli were isolated and successfully used for ratiometric confocal measurements with fluorescent dyes or patch‐clamp measurements. In conclusion, this rat model shows the features of diabetic kidney injury and is a good basis for the assessment of calcium signaling and glomerular filtration barrier function during type 1 DN.

The inward‐rectifying channels play an important role in the control of resting membrane potential and tubular homeostasis in the kidney. To illuminate the importance of Kcnj16 (Kir 5.1) in the context of a disease state in vivo, we generated a Kcnj16 knockout rat model in Dahl salt‐sensitive (SS) background by using zinc finger nuclease (ZFN) technology. ZFN against Kcnj16 caused a 18‐bp in‐frame deletion that occurred in the second protein transmembrane domain. IHC analysis demonstrated highly specific expression of Kcnj16 on the basolateral membranes of cortical collecting ducts and distal convoluted tubules in the control kidneys of SS and Sprague Dawley rats, which was completely abolished in Kcnj16‐/‐ rats; thus, successful knock out of this protein and consequent degradation of the channel in renal tubules were shown. The mean arterial pressure was significantly lower in Kcnj16‐/‐ compared to wild type SS rats (91.3±1.8 to 104.7±5.5 mmHg) when animals were fed a low salt (0.4%) diet. BUN, potassium and magnesium plasma concentrations were significantly modulated in knock out rats. Urea electrolyte balance was also disturbed compared to wild type animals. Importantly, change of the diet to high salt (4%) chow resulted in mortality of KO rats within 1‐2 days. These data demonstrate critical role of Kcnj16 channels in renal salt handling and in the development of salt‐sensitive hypertension. Grant Funding Source: Supported by NIH HL108880 and ADA 1‐10‐BS‐168 to A.S., NHLBI 5RC2HL101681 to H.J.J