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. 2015 Dec 10:5:17637.
doi: 10.1038/srep17637.

Podocyte injury in diabetic nephropathy: implications of angiotensin II-dependent activation of TRPC channels

Daria V Ilatovskaya  1 , Vladislav Levchenko  1 , Andrea Lowing  1 , Leonid S Shuyskiy  1   2 , Oleg Palygin  1 , Alexander Staruschenko  1   3
Affiliations

Podocyte injury in diabetic nephropathy: implications of angiotensin II-dependent activation of TRPC channels

Daria V Ilatovskaya et al. Sci Rep. .

Abstract

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.

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Figures

Figure 1
Figure 1. Development of STZ-induced diabetic nephropathy in Dahl SS rats during the 11 week long protocol.
(a) Experimental protocol used for type 1 diabetes induction in the Dahl SS rat. (b) Development of diabetes in SS rats during 11 weeks after STZ injection as monitored by blood glucose level, body weight and urine output changes. The red and green arrows denote the time of STZ injection and subsequent insulin pellet implantation, respectively. N = 8 rats in control and 9 rats in the STZ-SS group. Asterisk denotes statistically significant difference from control values (p < 0.05).
Figure 2
Figure 2. Kidney injury evaluation, microalbuminuria and nephrinuria in the course of DN development in STZ-treated SS rats.
All measurements were taken during the 11 week long protocol. (a) Histological changes indicative of DN progression in the kidney tissue of STZ-SS rats compared to control animals. Shown are cortical fragments of the trichrome stained kidneys at 10 x and 40 x magnifications. (b) Glomerular injury quantification; at least 4 rats and 100 glomeruli per rat were evaluated in each group. Asterisk denotes difference from the corresponding value in control rats, p < 0.05. (c) Microalbuminuria, nephrinuria and urinary creatinine in STZ- SS rats versus control animals; urine samples were taken for analysis 3 days before STZ injection, on day 7 after STZ injection (day of implant administration), day 42 (except for nephrin measurement), and day 77 (terminal point). Asterisks located above the data points in the STZ-treated group denote difference from the corresponding value in control group, p < 0.05. N = 8 rats in control and 9 rats in STZ-injected group.
Figure 3
Figure 3. Basal intracellular calcium concentration is higher in the podocytes of the diabetic animals.
(a) Representative calcium imaging traces showing a typical experiment designed to assess the intracellular calcium level in the podocytes. To measure intracellular calcium concentration, glomeruli were loaded with Fluo-4, AM, fluorescence intensity was recorded in the baseline and after addition of ionomycin and MnCl2. The graph demonstrates the fluorescence signal changes in response to ionomycin (producing the maximum of the Fluo-4,AM fluorescence, Fmax) and MnCl2, which quenches the dye and results in the lowest fluorescence intensity (Fmin). Intensity of fluorescence (left axis) for each time point was translated into the actual calcium concentration in nanomoles (right axis) according to the formula shown on the graph. The transients shown on the graph reflect fluorescence intensity of representative ROIs selected from glomeruli of a diabetic and control rat; images were taken every 4 s. (b) Bar graph summarizing the concentration of intracellular calcium in the podocytes of diabetic and control SS rats in the absence of any stimuli. Asterisk denotes statistically significant difference between groups (p < 0.05). Number of podocytes analyzed in each group (n) is shown on the graph.
Figure 4
Figure 4. Ang II-stimulated calcium influx in the podocytes of STZ-treated and control rats.
(a) Representative images of the Fluo-4,AM loaded glomeruli of control (upper row) and diabetic (lower row) SS rats before application of Ang II and at the point of maximal calcium influx after addition of the drug. Fluo-4,AM fluorescence intensity is positively related to calcium concentration within the podocytes. (b) Upper row demonstrates representative transients of intracellular calcium dynamics in the podocytes of the Fluo-4,AM loaded control and STZ-SS rat glomeruli. Low row shows the bar graph summarizing the relative changes in intracellular calcium concentration in response to Ang II in STZ-treated and control animals (reflected by Fluo-4,AM intensity). Number of podocytes for each group (n) is shown, asterisk indicates p < 0.05. N of rats used for these measurements was 7 in control and 6 in STZ-injected group.
Figure 5
Figure 5. Activity of TRPC6 channels in response to Ang II in type 1 diabetes.
(a) A representative patch-clamp recording showing the activation of TRPC channels in the podocytes of the control (vehicle-treated) SS rats in response to application of 1 μM Ang II, followed by a washout of Ang II. c and oi denote closed and open states of the channel, respectively; a full recording (upper row) and a fragment of a recording at a larger scale are shown. The recording was obtained at −60 mV. (b) Right panel demonstrates a summary graph of the open probability (Po) of the TRPC channels recorded in the podocytes of the STZ-treated animals compared to control rats after acute stimulation with 1 μM of Ang II. Representative current traces illustrating TRPC channels’ activity in podocytes of diabetic rats vs. control animals (before and after application of Ang II) are shown on the left panel. N represents number of animals studied, and n is the number of analyzed patch-clamp recordings; the recordings were obtained at −60 mV. * and # denote statistical significance (P < 0.05).
Figure 6
Figure 6. Expression levels of TRPC6 channels in the cortex of STZ-treated and control rats.
(a) A representative Western blot and a summary graph for the densitometry values of the TRPC6 channel signal blotted from the cortical kidney lysates of the control and STZ-treated rats (terminal point, 11 weeks after STZ injection). Loading control for the representative blot (β-actin) and number of animals per group analyzed in the summary graph (N) are shown. Asterisk denotes statistical significance (P < 0.05). (b) Images of the representative immunohistochemical stainings for TRPC6 in the control and STZ-treated rats. Magnification – 20 x; scale bar is shown.
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