Chronic kidney disease–mineral bone disorder (CKD‐MBD) is defined by abnormalities in mineral and hormone metabolism, bone histomorphometric changes, and/or the presence of soft‐tissue calcification. Emerging evidence suggests that features of CKD‐MBD may occur early in disease progression and are associated with changes in osteocyte function. To identify early changes in bone, we utilized the jck mouse, a genetic model of polycystic kidney disease that exhibits progressive renal disease. At 6 weeks of age, jck mice have normal renal function and no evidence of bone disease but exhibit continual decline in renal function and death by 20 weeks of age, when approximately 40% to 60% of them have vascular calcification. Temporal changes in serum parameters were identified in jck relative to wild‐type mice from 6 through 18 weeks of age and were subsequently shown to largely mirror serum changes commonly associated with clinical CKD‐MBD. Bone histomorphometry revealed progressive changes associated with increased osteoclast activity and elevated bone formation relative to wild‐type mice. To capture the early molecular and cellular events in the progression of CKD‐MBD we examined cell‐specific pathways associated with bone remodeling at the protein and/or gene expression level. Importantly, a steady increase in the number of cells expressing phosphor‐Ser33/37‐β‐catenin was observed both in mouse and human bones. Overall repression of Wnt/β‐catenin signaling within osteocytes occurred in conjunction with increased expression of Wnt antagonists (SOST and sFRP4) and genes associated with osteoclast activity, including receptor activator of NF‐κB ligand (RANKL). The resulting increase in the RANKL/osteoprotegerin (OPG) ratio correlated with increased osteoclast activity. In late‐stage disease, an apparent repression of genes associated with osteoblast function was observed. These data confirm that jck mice develop progressive biochemical changes in CKD‐MBD and suggest that repression of the Wnt/β‐catenin pathway is involved in the pathogenesis of renal osteodystrophy. © 2012 American Society for Bone and Mineral Research.

The incidence of cardiovascular events and mortality strongly correlates with serum phosphate in individuals with CKD. The Npt2b transporter contributes to maintaining phosphate homeostasis in the setting of normal renal function, but its role in CKD-associated hyperphosphatemia is not well understood. Here, we used adenine to induce uremia in both Npt2b-deficient and wild-type mice. Compared with wild-type uremic mice, Npt2b-deficient uremic mice had significantly lower levels of serum phosphate and attenuation of FGF23. Treating Npt2b-deficient mice with the phosphate binder sevelamer carbonate further reduced serum phosphate levels. Uremic mice exhibited high turnover renal osteodystrophy; treatment with sevelamer significantly decreased the number of osteoclasts and the rate of mineral apposition in Npt2b-deficient mice, but sevelamer did not affect bone formation and rate of mineral apposition in wild-type mice. Taken together, these data suggest that targeting Npt2b in addition to using dietary phosphorus binders may be a therapeutic approach to modulate serum phosphate in CKD.

Alterations to the structure of the glomerular filtration barrier lead to effacement of podocyte foot processes, leakage of albumin, and the development of proteinuria. To better understand the signaling pathways involved in the response of the glomerular filtration barrier to injury, we studied freshly isolated rat glomeruli, which allows for the monitoring and pharmacologic manipulation of early signaling events. Administration of protamine sulfate rapidly damaged the isolated glomeruli, resulting in foot process effacement and albumin leakage. Inhibition of calcium channels and chelation of extracellular calcium reduced protamine sulfateinduced damage, suggesting that calcium signaling plays a critical role in the initial stages of glomerular injury. Calcineurin inhibitors (FK506 and cyclosporine A) and the cathepsin L inhibitor E64 all inhibited protamine sulfate-mediated barrier changes, which suggests that calcium signaling acts, in part, through calcineurin-and cathepsin L-dependent cleavage of synaptopodin, a regulator of actin dynamics. The mTOR inhibitor rapamycin also protected glomeruli, demonstrating that calcium signaling has additional calcineurinindependent components. Furthermore, activation of Akt through mTOR had a direct role on glomerular barrier integrity, and activation of calcium channels mediated this process, likely independent of phosphoinositide 3-kinase. Taken together, these results demonstrate the importance of calcium and related signaling pathways in the structure and function of the glomerular filtration barrier.

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The weaning to estrus and weaning to ovulation intervals in sows are controlled by ovarian follicular growth after weaning. Longer intervals could be caused by smaller diameter follicles at weaning that take more time to reach a preovulatory size. We addressed this hypothesis by decreasing the diameter of follicular populations before weaning and then measuring follicular development and interval to estrus and ovulation after weaning. The posterior vena cava, cranial to the entry of the ovarian vein, was cathetered for blood sampling and infusion in 20 sows at 12 +/- 1 d after farrowing. Sows were assigned randomly to receive either 30 mL of charcoal-treated follicular fluid (FF, n = 9; a treatment known to decrease serum FSH and follicular diameter) or 30 mL of saline (n = 11) by venous infusion thrice daily (0700, 1500, and 2300 h) for 96 h beginning at 14 +/- 1 d after farrowing. Sows were weaned 48 h after the last infusion. Blood samples were collected for FSH analysis thrice daily beginning on the day of catheterization and continuing until ovulation. Follicular diameter was determined once daily by transrectal ultrasonography. A treatment x time interaction was detected for serum FSH (P < 0.001) and follicular diameter (P < 0.001) because serum FSH and the diameter of follicular populations decreased in FF sows during the infusion period. After the infusion period, serum FSH rebounded in FF sows, and follicles resumed growth but grew at the same rate as those of saline-treated sows, thus failing to achieve equivalent diameters relative to saline-treated sows on a given day after weaning. As a result, sows treated with FF had longer (P < 0.05) weaning to estrus (6.1 +/- 0.4 d) and weaning to ovulation (8.6 +/- 0.5 d) intervals compared with saline-treated sows (4.7 +/- 0.4 d and 7.2 +/- 0.4 d, respectively). We conclude that the diameter of the follicular population at weaning is one factor that controls interval to estrus and ovulation in sows. Small follicles at weaning cannot undergo compensatory growth and require additional time to reach a preovulatory size.

Polycystic kidney diseases (PKDs) are genetic diseases characterized by renal cyst formation with increased cell proliferation, apoptosis, and transition to a secretory phenotype at the expense of terminal differentiation. Despite recent progress in understanding PKD pathogenesis and the emergence of potential therapies, the key molecular mechanisms promoting cystogenesis are not well understood. Here, we demonstrate that mechanisms including endoplasmic reticulum stress, oxidative damage, and compromised mitochondrial function all contribute to nephronophthisis-associated PKD. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is emerging as a critical mediator of these cellular processes. Therefore, we reasoned that pharmacological targeting of CaMKII may translate into effective inhibition of PKD in jck mice. Our data demonstrate that CaMKII is activated within cystic kidney epithelia in jck mice. Blockade of CaMKII with a selective inhibitor results in effective inhibition of PKD in jck mice. Mechanistic experiments in vitro and in vivo demonstrated that CaMKII inhibition relieves endoplasmic reticulum stress and oxidative damage and improves mitochondrial integrity and membrane potential. Taken together, our data support CaMKII inhibition as a new and effective therapeutic avenue for the treatment of cystic diseases.

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GH receptor (GHR) messenger RNA (mRNA) is transcribed from at least three different promoters within the liver of cattle. The first promoter (P1) is liver specific and alternatively splices exon 1A onto the GHR mRNA (GHR 1A mRNA). The second and third promoters (P2 and P3) have constitutive activity in many tissues and alternatively splice exons 1B and 1C onto the GHR mRNA (GHR 1B and GHR 1C mRNA). The total amount of GHR in the liver partially determines liver insulin-like growth factor I (IGF-I) synthesis in response to GH. Two studies were conducted to characterize the changes in GHR 1A mRNA, alternatively spliced GHR mRNA, and IGF-I mRNA during late pregnancy and early lactation in dairy cattle. Liver RNA was isolated from pregnant Holstein cattle (Bos taurus) on days -14, 0, and 21 relative to parturition (study 1) or days -14, 0, 15, 30, 60, and 90 relative to parturition (study 2). Ribonuclease protection assays were used to quantify total GHR (all GHR variants) as well as liver-specific GHR 1A and alternatively spliced GHR mRNA. Likewise, total IGF-I as well as alternatively spliced IGF-I mRNA (class 1 and class 2 transcripts) were measured. A decrease in total GHR mRNA at parturition (P < 0.01) was associated with a specific decrease in GHR 1A mRNA (P < 0.001). The amount of alternatively spliced GHR mRNA (including GHR 1B and GHR 1C mRNA) did not change at parturition (P > 0.10). Total liver IGF-I mRNA and blood IGF-I concentrations were also decreased at parturition (P < 0.05 and P < 0.01, respectively). However, a decrease in IGF-I mRNA was observed for both class 1 and class 2 IGF-I transcripts (P < 0.01 and P < 0.05, respectively). We conclude that the reduced amount of GHR mRNA during early lactation is caused by a specific down-regulation of GHR 1A mRNA that was associated with decreased liver IGF-I mRNA and decreased blood IGF-I concentrations. These data provide evidence for independent regulation of GHR mRNA by mechanisms that discriminate between GHR P1 (transcribes GHR 1A) and alternative promoters that transcribe constitutive GHR mRNA.