The Role of Nitric Oxide in the Dysregulation of the Urine Concentration Mechanism in Diabetes Mellitus

Uncontrolled diabetes mellitus results in osmotic diuresis. Diabetic patients have lowered nitric oxide (NO) which may exacerbate polyuria. We examined how lack of NO affects the transporters involved in urine concentration in diabetic animals. Diabetes was induced in rats by streptozotocin. Control and diabetic rats were given L-NAME for 3 weeks. Urine osmolality, urine output, and expression of urea and water transporters and the Na-K-2Cl cotransporter were examined. Predictably, diabetic rats presented with polyuria (increased urine volume and decreased urine osmolality). Although metabolic parameters of control rats were unaffected by L-NAME, treated diabetic rats produced 30% less urine and osmolality was restored. UT-A1 and UT-A3 were significantly increased in diabetic-rat inner medulla. While L-NAME treatment alone did not alter UT-A1 or UT-A3 abundance, absence of NO prevented the upregulation of both transporters in diabetic rats. Similarly, AQP2 and NKCC2 abundance was increased in diabetic animals however, expression of these transporters were unchanged by L-NAME treatment of diabetes. Increased expression of the concentrating transporters observed in diabetic rats provides a compensatory mechanism to decrease solute loss despite persistent glycosuria. Our studies found that although diabetic-induced glycosylation remained increased, total protein expression was decreased to control levels in diabetic rats treated with L-NAME. While the role of NO in urine concentration remains unclear, lowered NO associated with diabetes may be deleterious to the transporters’ response to the subsequent osmotic diuresis

Absence of PKC-alpha attenuates lithium-induced nephrogenic diabetes insipidus.

Lithium, an effective antipsychotic, induces nephrogenic diabetes insipidus (NDI) in ∼40% of patients. The decreased capacity to concentrate urine is likely due to lithium acutely disrupting the cAMP pathway and chronically reducing urea transporter (UT-A1) and water channel (AQP2) expression in the inner medulla. Targeting an alternative signaling pathway, such as PKC-mediated signaling, may be an effective method of treating lithium-induced polyuria. PKC-alpha null mice (PKCα KO) and strain-matched wild type (WT) controls were treated with lithium for 0, 3 or 5 days. WT mice had increased urine output and lowered urine osmolality after 3 and 5 days of treatment whereas PKCα KO mice had no change in urine output or concentration. Western blot analysis revealed that AQP2 expression in medullary tissues was lowered after 3 and 5 days in WT mice; however, AQP2 was unchanged in PKCα KO. Similar results were observed with UT-A1 expression. Animals were also treated with lithium for 6 weeks. Lithium-treated WT mice had 19-fold increased urine output whereas treated PKCα KO animals had a 4-fold increase in output. AQP2 and UT-A1 expression was lowered in 6 week lithium-treated WT animals whereas in treated PKCα KO mice, AQP2 was only reduced by 2-fold and UT-A1 expression was unaffected. Urinary sodium, potassium and calcium were elevated in lithium-fed WT but not in lithium-fed PKCα KO mice. Our data show that ablation of PKCα preserves AQP2 and UT-A1 protein expression and localization in lithium-induced NDI, and prevents the development of the severe polyuria associated with lithium therapy

AQP2 expression is not changed in short-term lithium-treated PKCα KO mice.

<p>A) IM tissue collected from injected WT and PKCα KO mice was subjected to Western blot analysis and probed for AQP2. Representative blots showing both nonglycosylated (29-kDa) and glycosylated (35- to 50-kDa) AQP2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101753#pone.0101753-Terris1" target="_blank">[59]</a>, and the corresponding loading control, β tubulin. Each lane represents one animal. B) Combined densitometry of all forms of AQP2 in the IM normalized to β tubulin. C) OM tissue probed for AQP2 and β tubulin. D) Combined densitometry of all forms of AQP2 in the OM normalized to β tubulin. Data are presented as percent difference in expression from Day 0, untreated mice as mean ± SEM where *  =  p<0.05 vs. WT day 0 and §  =  p<0.05 vs. PKCα KO day 0 is deemed significant. <i>n = 6</i>.</p

UT-A1 expression is not changed in short-term lithium-treated PKCα KO mice.

<p>IM tissue collected from injected WT and PKCα KO mice was subjected to Western blot analysis and probed for UT-A1. A) Representative blots showing the multiple glycosylated forms of UT-A1 that span between 97 and 117 kDa <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101753#pone.0101753-Chen1" target="_blank">[60]</a>, and the corresponding loading control, β tubulin. Each lane represents one animal. B) Combined densitometry of all glycosylated forms of UT-A1 normalized to β tubulin. Data are presented as percent difference in expression from Day 0, untreated mice as mean ± SEM where *  =  p<0.05 vs. WT day 0 and §  =  p<0.05 vs. PKCα KO day 0 is deemed significant. <i>n = 6</i>.</p

UT-A1 expression is not altered in long-term lithium treatment PKCα KO mice.

<p>IM tissue collected from lithium-fed WT and PKCα KO mice was subjected to Western blot analysis and probed for UT-A1. A) Representative blots showing the multiple glycosylated forms of UT-A1 (bracketed) and corresponding loading control, β tubulin. Each lane represents one animal. B) Combined densitometry of all glycosylated forms of UT-A1 normalized to β tubulin. Data are presented as mean ± SEM where *  =  p<0.05 vs. WT day 0 and §  =  p<0.05 vs. PKCα KO day 0 is deemed significant. <i>n = 12</i>.</p

Lithium-induced NDI is attenuated in PKCα KO mice.

<p>WT and PKCα KO mice were provided standard chow or chow containing lithium (40 mmol/kg) for 6 weeks. Single animals were subsequently placed in individual metabolic cages to determine 24-h water intake. Urine and serum were also collected at this time point for metabolic determinations. Uprot/Uosm  =  urinary protein/urine osmolality ratio, CrCl/BW  =  creatinine clearance normalized to body weight (29–30 g). Data are presented as mean ± SEM where *  =  p<0.05 vs. WT day 0 and §  =  p<0.05 vs. PKCα KO day 0 is deemed significantly different. <i>n = 5</i>.</p

PKCα KO mice are resistant to lithium-induced natriuresis, kaliuresis and hypercalciuria.

<p>Urinary sodium (A), chloride (B), potassium (C) and calcium (D) were measured and normalized to urinary creatinine to examine difference between lithium-fed WT and PKCα KO to untreated control groups. Data are presented as mean ± SEM where *  =  p<0.05 untreated vs. 6 week-fed lithium treatment. <i>n = 12.</i></p

PKCα KO mice do not develop polyuria in response to short-term lithium treatment.

<p>PKCα KO mice and littermate controls (WT) were injected daily with 40 mmol/kg of lithium for 3 or 5 consecutive days. Urine was collected via metabolic cages and urine osmolality was determined. Data are presented as mean ± SEM where *  =  p<0.05 vs. WT day 0 and §  =  p<0.05 vs. PKCα KO day 0 is deemed significant. <i>n = 6</i>.</p

Acute lithium treatment does not induce NDI in PKCα KO mice.

<p>PKCα KO and WT mice were injected intraperitoneally with 40 mmol/kg LiCl in saline every 24 hours up to 3 or 5 days. Single animals were subsequently placed in individual metabolic cages and urine and serum were collected after 0, 3 or 5 days of daily lithium treatments. Uprot/Uosm  =  urinary protein/urine osmolality ratio, CrCl/BW  =  creatinine clearance normalized to body weight (23–25 g). Data are presented as mean ± SEM where *  =  p<0.05 vs. WT day 0 and §  =  p<0.05 vs. PKCα KO day 0 is deemed significantly different. <i>n</i> = 6.</p