LA-induced mitochondrial Ca²⁺ efflux was responsible for peroxynitrite generation.

<p>Mitochondria were prepared from HM cells and labeled with X-rhod-1 (2 µM) and DCF (1 µM) with (A) or without Ca²⁺ (B). LA-induced mitochondrial Ca²⁺ efflux (black traces) and peroxynitrite generation (red traces) were indexed by the gradual decrease in X-rhod-1 and increase in DCF fluorescence intensity, respectively.</p

Mechanism of excessive NEFA contributing to pathogenesis of diabetic nephropathy.

<p>NEFA, such as linoleic acid (LA, an 18∶2 n-6 polyunsaturated fatty acid) induces Ca²⁺ efflux from mitochondria by activating a hsp90β1-dependent pathway (PIMCE). The LA responsive mitochondrial Ca²⁺ efflux diminishes the inhibitory effect of Ca²⁺ on superoxide production from complexes I or III and enhances nitrite conversion to NO by complex III, resulting in increased peroxynitrite formation and protein nitrotyrosylation. Protein nitrotyrosylation may disrupt the normal functions of mesangial cells and the kidney, leading to abnormalities in the structure and/or function of diabetic kidney.</p

LA-induced mitochondrial Ca²⁺ efflux and protein nitrotyrosylation were enhanced in the kidney of db/db mice.

<p>Mitochondria were prepared from fresh kidney tissues of 12–16 wk old db/+ and db/db mice and labeled with X-rhod-1 (2 µM). (A): LA-induced mitochondrial Ca²⁺ efflux in the kidney of db/+ and db/db mice. (B): The ratio of the initial rate of LA-induced mitochondrial Ca²⁺ efflux (V_LA; measured in the first 60 s following LA) to the basal rate (V₀; measured prior to LA). The values represent mean±SEM of V_LA/V₀, **<i>P</i><0.01, n = 3. (C) and (D): Nitrotyrosine levels in the kidney homogenates of four pairs of db/+ and db/db mice were analyzed by western blot using α-tubulin as loading control. The arrowhead highlights a protein band (∼22 kDa) with enhanced nitrotyrosine levels in db/db mice, **<i>P</i><0.01, n = 4. (E) and (F): Representatives of the nitrotyrosine levels in the kidney sections of db/+ and db/db mice as determined by immunohistochemistry.</p

Mitochondrial complexes I and III were required for LA-induced peroxynitrite generation.

<p>HM cells were labeled with fura-2 and DCF to measure LA responsive [Ca²⁺]_i mobilization (black traces) and peroxynitrite generation (red traces). (A): LA responses in untreated cells (control); (B) LA responses in cells pretreated with rotenone (a mitochondrial complex I inhibitor, 10 µM, 20 min); (C) and (D): LA responses in cells pretreated with the mitochondrial complex III blockers, antimycin A (1 µM, 20 min; C) or myxothiazol (0.5 µM, 60 min; D).</p

The effect of hsp90β1 on LA-induced [Ca²⁺]_i mobilization and peroxynitrite generation.

<p>HM cells were treated with vehicle (control), hsp90β1 RNAi, or 17-DMAG for 48 h. (A): Cell lysates were prepared and the alterations of hsp90β1 expression were analyzed by western blot analysis using α-tubulin as loading control. (B): The alteration of LA-induced [Ca²⁺]_i mobilization and peroxynitrite generation in hsp90β1 RNAi- and 17-DMAG-treated HM cells relative to control. The values represent the mean±SEM. *<i>P</i><0.05, **<i>P</i><0.01, n = 6.</p

Mitochondria played an essential role in LA-induced [Ca²⁺]_i mobilization and peroxynitrite generation.

<p>HM cells were labeled with fura-2 and DCF to measure [Ca²⁺]_i mobilization (black traces) and peroxynitrite generation (red traces). (A): Bradykinin (BK, 100 nM) responses; (B): thapsigargin (TG, 2 µM) responses; (C) and (F): LA responses measured in the presence of extracellular Ca²⁺ (control); (D): LA responses measured without extracellular Ca²⁺; (G) LA responses measured in cells pretreated with BAPTA (50 µM, 30 min) without extracellular Ca²⁺. In the absence of extracellular Ca²⁺, (I): LA responses (control); (J): LA responses in cells pretreated with TG (2 µM, 5 min); (K) LA responses in cells pretreated with FCCP (a mitochondrial uncoupler; 4 µM, 5 min). The values in graphs (E, H, and L) represent the mean±SEM of the relative amplitudes of LA-induced [Ca²⁺]_i and peroxynitrite responses, *<i>P</i><0.05, **<i>P</i><0.01, n = 6, treated cells <i>vs</i> control.</p

Impact of 17-DMAG on hsp90β1, [Ca²⁺]_m, and peroxynitrite generation in the kidney.

<p>Western blot analysis was performed to assess hsp90β1 in kidney homogenate (<i>A</i>) and isolated mitochondria (<i>B</i>) of HFD-fed <i>db/db</i> mice with 6 animals per group. Linoleic acid (LA)-induced [Ca²⁺]_m efflux and peroxynitrite generation (<i>C–F</i>) in kidney mitochondria were measured.</p

Kidney histopathology of HFD-fed <i>db/db</i> mice.

<p>Representative figures showing the photomicrographs of HE (<i>A–F</i>), PAS (<i>G</i>, <i>H</i>), and Masson's trichrome stained sections from <i>db/db-HF-S</i> group (<i>C, E, G, I</i>) and <i>db/db-HF-G</i> group (<i>D, F, H, J</i>) taken at 200× magnification. Image-based computer assisted analysis was performed to quantify tubular damage index (<i>K</i>), mesangial expansion (<i>L</i>), and interstitial collagen accumulation (<i>M</i>) from 6 animals per group. (A) and (B) showed parallel experiments with HFD-fed <i>db/+</i> mice injected with saline and 17-DMAG, respectively.</p

Effect of 17-DMAG on survival rate of HFD-fed <i>db/db</i> mice.

<p>Kaplan-Meyer survival analysis was performed using the log-rank statistics to measure the difference between the survival curves of d<i>b/db-HF-S</i> vs <i>db/db-HF-G</i> mice with n = 9 per group. Parallel experiments were performed with <i>db/+</i> mice (<i>db/+-HF-S</i> and <i>db/+-HF-G</i> groups) and no mortality was observed.</p

Effects of high fat diet (HFD) and 17-DMAG treatment on kidney function of <i>db/db</i> mice.

<p>(<i>A</i>) Schematic of two phases of HFD feeding and 17-DMAG treatment with the arrows indicating the scheduled kidney function assessments. During the first phase of HFD and the subsequent regular diet (RD) feeding, the 24 h urinary albumin excretion (<i>B</i>) and urine output (<i>C</i>) were measured to assess kidney functions in <i>db/db</i> and the non-diabetic control (<i>db/+</i>) mice. During the second phase of HFD feeding, the animals were either injected with saline (<i>HF-S</i>) or 17-DMAG (<i>HF-G</i>) and the kidney functions were initially assessed by the 24 h urinary albumin excretion (<i>D</i>) and urine output (<i>E</i>), and then by serum creatinine (<i>G</i>) when anuria or oliguria occurred. Blood glucose was measured as indicated in (<i>F)</i>. **<i>P</i><0.01, compared with baseline assessed on day 0 with n = 6–12 per group.</p