Rachel W. Fallet

Potassium channel contributions to afferent arteriolar tone in normal and diabetic rat kidney

We previously reported an enhanced tonic dilator impact of ATP-sensitive K+ channels in afferent arterioles of rats with streptozotocin (STZ)-induced diabetes. The present study explored the hypothesis that other types of K+ channel also contribute to afferent arteriolar dilation in STZ rats. The in vitro blood-perfused juxtamedullary nephron technique was utilized to quantify afferent arteriolar lumen diameter responses to K+ channel blockers: 0.1-3.0 mM 4-aminopyridine (4-AP; KV channels), 10-100 microM barium (KIR channels), 1-100 nM tertiapin-Q (TPQ; Kir1.1 and Kir3.x subfamilies of KIR channels), 100 nM apamin (SKCa channels), and 1 mM tetraethylammonium (TEA; BKCa channels). In kidneys from normal rats, 4-AP, TEA, and Ba2+ reduced afferent diameter by 23 +/- 3, 8 +/- 4, and 18 +/- 2%, respectively, at the highest concentrations employed. Neither TPQ nor apamin significantly altered afferent diameter. In arterioles from STZ rats, a constrictor response to TPQ (22 +/- 4% decrease in diameter) emerged, and the response to Ba2+ was exaggerated (28 +/- 5% decrease in diameter). Responses to the other K+ channel blockers were similar to those observed in normal rats. Moreover, exposure to either TPQ or Ba2+ reversed the afferent arteriolar dilation characteristic of STZ rats. Acute surgical papillectomy did not alter the response to TPQ in arterioles from normal or STZ rats. We conclude that 1) KV, KIR, and BKCa channels tonically influence normal afferent arteriolar tone, 2) KIR channels (including Kir1.1 and/or Kir3.x) contribute to the afferent arteriolar dilation during diabetes, and 3) the dilator impact of Kir1.1/Kir3.x channels during diabetes is independent of solute delivery to the macula densa.

Tyrosine Kinase Involvement in Renal Arteriolar Constrictor Responses to Angiotensin II

Experiments were performed to test the hypothesis that tyrosine kinase activity contributes to renal arteriolar contractile responses to angiotensin (Ang) II. Rats were subjected to short-term enalaprilat treatment to decrease endogenous Ang II formation before tissue was harvested for experiments with the in vitro blood-perfused juxtamedullary nephron technique. Acute surgical papillectomy was used to avoid the indirect afferent arteriolar effect of Ang II that arises through increased tubuloglomerular feedback sensitivity. Arteriolar lumen diameter responses to 1 and 10 nmol/L Ang II were monitored by videomicroscopic methods before and during treatment with various tyrphostin compounds: 100 &mgr;mol/L AG18 (broad-spectrum tyrosine kinase inhibitor), 100 nmol/L AG1478 (selective epidermal growth factor receptor tyrosine kinase inhibitor), or 100 &mgr;mol/L AG9 (inactive analog). Baseline afferent arteriolar lumen diameter averaged 23.5±1.2 &mgr;m and was not influenced by any tyrphostin. Ang II (10 nmol/L) decreased afferent diameter by 11.1±1.0 &mgr;m under untreated conditions, a response that was not altered by AG9 but significantly blunted by AG18 (34±9% inhibition) or AG1478 (52±8% inhibition). AG18 did not suppress afferent arteriolar contractile responses to membrane depolarization (20 to 55 mmol/L K+ bath). Efferent arteriolar baseline diameter averaged 24.1±0.8 &mgr;m and was unaltered by AG18 or AG1478; however, efferent diameter responses to 10 nmol/L Ang II were diminished 52±10% by AG18 and 51±13% by AG1478. These observations indicate that Ang II signaling in renal afferent and efferent arteriolar vascular smooth muscle is either mediated or modulated by tyrosine kinase activity, including that of the epidermal growth factor receptor tyrosine kinase.

Efficacy of phosphodiesterase-4 inhibitors in juvenile Batten disease (CLN3)

Juvenile neuronal ceroid lipofuscinosis (JNCL), or juvenile Batten disease, is a pediatric lysosomal storage disease caused by autosomal recessive mutations in CLN3, typified by blindness, seizures, progressive cognitive and motor decline, and premature death. Currently, there is no treatment for JNCL that slows disease progression, which highlights the need to explore novel strategies to extend the survival and quality of life of afflicted children. Cyclic adenosine monophosphate (cAMP) is a second messenger with pleiotropic effects, including regulating neuroinflammation and neuronal survival. Here we investigated whether 3 phosphodiesterase‐4 (PDE4) inhibitors (rolipram, roflumilast, and PF‐06266047) could mitigate behavioral deficits and cell‐specific pathology in the Cln3Δex7/8 mouse model of JNCL.

Caspase 1 activity influences juvenile Batten disease (CLN3) pathogenesis

Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) is an autosomal recessive lysosomal storage disease caused by loss‐of‐function mutations in CLN3. Symptoms appear between 5 and 10 years of age, beginning with blindness and seizures, followed by progressive cognitive and motor decline, and premature death. Glial activation and impaired neuronal activity are early signs of pathology in the Cln3Δex7/8 mouse model of JNCL, whereas neuron death occurs much later in the disease process. We previously reported that Cln3Δex7/8 microglia are primed toward a pro‐inflammatory phenotype typified by exaggerated caspase 1 inflammasome activation and here we extend those findings to demonstrate heightened caspase activity in the Cln3Δex7/8 mouse brain. Based on the ability of caspase 1 to cleave a large number of substrates that have been implicated in JNCL pathology, we examined the functional implications of caspase 1 inflammasome activity by crossing Cln3Δex7/8 and caspase 1‐deficient mice to create Cln3Δex7/8/Casp‐1−/− animals. Caspase 1 deletion influenced motor behavior deficits and astrocyte activation in the context of CLN3 mutation, since both were significantly reversed in Cln3Δex7/8/Casp‐1−/− mice, with phenotypes approaching that of wild‐type animals. We also report a progressive age‐dependent reduction in whisker length in Cln3Δex7/8 mice that was partially caspase 1‐dependent. However, not all CLN3 phenotypes were reversed following caspase 1 deletion, since no significant differences in lysosomal accumulation or microglial activation were observed between Cln3Δex7/8 and Cln3Δex7/8/Casp‐1−/− mice. Although the molecular targets of aberrant caspase 1 activity in the context of CLN3 mutation remain to be identified, our studies suggest that caspase 1 may represent a potential therapeutic target to mitigate some attributes of CLN3 disease.

Renoprotective impact of estrogen receptor α and its splice variants in female mice with type 1 diabetes

Estrogen has been implicated in the regulation of growth and immune function in the kidney, which expresses the full-length estrogen receptor-α (ERα66), its ERα splice variants, and estrogen receptor-β (ERβ). Thus, we hypothesized that these splice variants may inhibit the glomerular enlargement that occurs early in type 1 diabetes (T1D). T1D was induced by streptozotocin (STZ) injection in 8- to 12-wk-old female mice lacking ERα66 (ERα66KO) or all ERα variants (αERKO), and their wild-type (WT) littermates. Basal renal ERα36 protein expression was reduced in the ERα66KO model and was downregulated by T1D in WT mice. T1D did not alter ERα46 or ERβ in WT-STZ; however, ERα46 was decreased modestly in ERα66KO mice. Renal hypertrophy was evident in all diabetic mice. F4/80-positive immunostaining was reduced in ERα66KO compared with WT and αERKO mice but was higher in STZ than in Control mice across all genotypes. Glomerular area was greater in WT and αERKO than in ERα66KO mice, with T1D-induced glomerular enlargement apparent in WT-STZ and αERKO-STZ, but not in ERα66KO-STZ mice. Proteinuria and hyperfiltration were evident in ERα66KO-STZ and αERKO-STZ, but not in WT-STZ mice. These data indicate that ERα splice variants may exert an inhibitory influence on glomerular enlargement and macrophage infiltration during T1D; however, effects of splice variants are masked in the presence of the full-length ERα66, suggesting that ERα66 acts in opposition to its splice variants to influence these parameters. In contrast, hyperfiltration and proteinuria in T1D are attenuated via an ERα66-dependent mechanism that is unaffected by splice variant status.