Aims/hypothesis The unfolded protein response (UPR) in endoplasmic reticulum (ER) and autophagy are known to be related. We investigated the role of autophagy in UPR of pancreatic beta cells and the susceptibility of autophagydeficient beta cells to the ER stress that is implicated in the development of diabetes. Methods Rat insulin promoter (RIP)-Cre + ;autophagy-related 7 (Atg7) F/W mice were bred with ob/w mice to derive RIP-Cre + ;Atg7 F/F -ob/ob mice and to induce ER stress in vivo. GFP-LC3 + -ob/ob mice were generated to examine in vivo autophagic activity. Real-time RT-PCR was performed to study the expression of the genes of the UPR machinery. Proteolysis was assessed by determining release of incorporated radioactive leucine.Results Production of UPR machinery was reduced in autophagy-deficient beta cells, which was associated with diminished production of p85α and p85β regulatory subunits of phosphoinositide 3-kinase. Because of compromised UPR machinery, autophagy-deficient beta cells were susceptible to ER stressors in vitro. When mice with beta cell-specific autophagy deficiency, which have mild hyperglycaemia, were bred with ob/ob mice to induce ER stress in vivo, severe diabetes developed, which was accompanied by an increase in beta cell death and accumulation of reactive oxygen species. The increased demand for UPR present in obesity was unmet in autophagy-deficient beta cells. Autophagy level and autophagic activity were enhanced by lipid, while proteolysis was reduced. Conclusions/interpretation These results suggest that autophagy is important for intact UPR machinery and appropriate UPR in response to lipid injury that increases demand for UPR. Autophagy deficiency in pancreatic beta cells may contribute to the progression from obesity to diabetes.

At birth, the rat renal papilla has the structural composition of the mature inner stripe of the outer medulla. All loops of Henle have the configuration of short loops, and there are no ascending thin limbs. This study examines the role of apoptosis in the differentiation of the loop of Henle and the development of the ascending thin limb in the rat kidney. Kidneys of 20-day-old fetuses and 1-, 3-, 5-, 7-, 14-, and 21-day-old pups were preserved for immunohistochemistry and electron microscopy. Using a preembedding immunoperoxidase method, we identified thick ascending limbs by labeling with antibodies to the serotonin receptor, 5-HT1A, and descending thin limbs were identified by labeling with antibodies to aquaporin-1. Three methods were used to identify apoptotic cells as follows: 1) in situ nick end labeling using the ApopTag kit, 2) toluidine blue staining on plastic sections followed by etching, and 3) transmission electron microscopy. At birth, tubules with 5-HT1A immunoreactivity were present throughout the renal papilla, and there were no ascending thin limbs. From 1 to 14 days of age, staining for apoptosis was observed in numerous cells in the 5-HT1A-positive epithelium, beginning at the papillary tip and ascending to the border between outer and inner medulla. This was associated with transformation from a cuboidal to a squamous epithelium and subsequent disappearance of 5-HT1A immunostaining from the transformed cells. Electron microscopy confirmed the presence of apoptotic cells and phagocytosed apoptotic bodies in the thick ascending limb in the renal papilla. We conclude that the ascending thin limb is derived from the 5-HT1A-positive thick ascending limb by apoptotic deletion of thick ascending limb cells and transformation of the remaining tubule cells into the 5-HT1A-negative ascending thin limb.

In the developing rat kidney, both type A and type B intercalated cells are present throughout the medullary collecting duct (MCD), as well as the papillary surface epithelium. After birth, intercalated cells gradually disappear from the papillary surface epithelium and the terminal MCD, and type B cells disappear from the entire MCD. The purpose of this study was to establish the mechanism(s) by which intercalated cells are deleted from the MCD during development. Kidneys from 14-, 16-, 18-, and 20-day-old fetuses and 1-, 3-, 7-, and 14-day-old pups were preserved for light microscopic immunohistochemistry and electron microscopy. Intercalated cells were identified by immunostaining for H(+)-adenosinetriphosphatase (H(+)-ATPase) and band 3 protein. Apoptosis was identified by nick end labeling of DNA fragments, staining with the vital dye toluidine blue, and transmission electron microscopy. Two distinct mechanisms of elimination of intercalated cells were detected. Cells with apical labeling for H(+)-ATPase and basolateral labeling for band 3 protein protruded into the lumen of the MCD as if they were being extruded from the epithelium, and many had lost contact with the basement membrane. Extrusion of the cells with basolateral H(+)-ATPase or with no labeling for H(+)-ATPase was never observed. Apoptosis was observed in the MCD from shortly before birth to 7 days after birth, gradually progressing from the papillary tip toward the outer medulla. Staining for apoptosis was present in H(+)-ATPase-positive apoptotic bodies, located in cells that were negative for H(+)-ATPase. Staining was also occasionally observed in apoptotic cells with basolateral H(+)-ATPase but never in cells with apical H(+)-ATPase. Electron microscopy confirmed the presence of apoptotic intercalated cells in the MCD and demonstrated that apoptotic bodies were located in inner medullary collecting duct (IMCD) cells and principal cells. These results demonstrate that intercalated cells are deleted from the MCD by two distinct mechanisms, one involving apoptosis and subsequent phagocytosis by neighboring principal cells or IMCD cells. Elimination by extrusion affects only type A intercalated cells, whereas deletion by apoptosis appears to occur only in type B intercalated cells.

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