During the past several years, sites of expression of ion transport proteins in tubules from adult kidneys have been described and correlated with functional properties. Less information is available concerning sites of expression during tubule morphogenesis, although such expression patterns may be crucial to renal development. In the current studies, patterns of renal axial differentiation were defined by mapping the expression of sodium transport pathways during nephrogenesis in the rat. Combined in situ hybridization and immunohistochemistry were used to localize the Na-Pi cotransporter type 2 (NaPi2), the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), the thiazide-sensitive Na-Cl cotransporter (NCC), the Na/Ca exchanger (NaCa), the epithelial sodium channel (rENaC), and 11β-hydroxysteroid dehydrogenase (11HSD). The onset of expression of these proteins began in post-S-shape stages. NKCC2 was initially expressed at the macula densa region and later extended into the nascent ascending limb of the loop of Henle (TAL), whereas differentiation of the proximal tubular part of the loop of Henle showed a comparatively retarded onset when probed for NaPi2. The NCC was initially found at the distal end of the nascent distal convoluted tubule (DCT) and later extended toward the junction with the TAL. After a period of changing proportions, subsegmentation of the DCT into a proximal part expressing NCC alone and a distal part expressing NCC together with NaCa was evident. Strong coexpression of rENaC and 11HSD was observed in early nascent connecting tubule (CNT) and collecting ducts and later also in the distal portion of the DCT. Ontogeny of the expression of NCC, NaCa, 11HSD, and rENaC in the late distal convolutions indicates a heterogenous origin of the CNT. These data present a detailed analysis of the relations between the anatomic differentiation of the developing renal tubule and the expression of tubular transport proteins.

The Han:SPRD rat model for inherited polycystic kidney disease (PKD) was characterized (clinical parameters, morphology, immunohistochemistry and in situ hybridization). Homozygous animals died of uremia after three to four weeks with severe cystic transformation of virtually all nephrons and collecting ducts (serum urea: 616 +/- 195 mg/dl; kidney-to-body weight ratio: > 20%). In heterozygotes, slow progression of the disease led to death between the 12th and 21st month (median: 17 months; serum urea levels above 200 mg/dl). Kidney enlargement was moderate, and cysts were restricted to the cortex and outer medulla. Immunohistochemical markers showed that approximately 75% of the cysts were derived from the proximal tubule. Cystic transformation started in the proximal tubule with a sharp onset of basement membrane alteration and a loss of epithelial differentiation restricted to small focal areas. In these areas, alpha 1(IV) collagen and laminin B1 mRNA were enhanced as revealed by isotopic and non-isotopic in situ hybridization. Fibroblasts underlying the affected tubular portions were involved in matrix overexpression resulting in subepithelial accumulation of immunoreactive collagen IV and laminin. In later stages of cystic transformation distal nephron segments were affected as well. A reversal in epithelial polarity as judged from Na,K-ATPase-immunoreactivity was not observed. Renal immunoreactive renin-status was significantly decreased. Hematocrit was lowered in heterozygotes (40.4 +/- 5.8 vol% compared to 46.7 +/- 1.99 vol% in controls; P < 0.05) and total renal EPO mRNA was reduced to 36 +/- 14% of the mean value of control animals, whereas serum EPO levels were not significantly altered. We conclude that the Han:SPRD rat is a useful model for the study of human ADPKD since both diseases are similar in several aspects. The model is particularly suitable for the study of epithelial-mesenchymal interactions at the beginning of tubular cystic transformation.

Aims/hypothesis. Insulin-producing beta cells are destroyed by oxidative and nitrosative stress during the pathogenesis of Type I (insulin-dependent) diabetes mellitus. These cells are more sensitive than others due to their deficiency of well known antioxidant enzymes like superoxide dismutase, glutathione peroxidase and catalase. However the peroxiredoxins discovered in the past decade form a large family of highly conserved thioredoxin-dependent peroxide reductases, which are present in most tissues. We investigated whether peroxiredoxins I and II are present in pancreatic beta cells and if they are inducible by oxidative and nitrosative stress. Methods. To detect these enzymes in insulin-producing beta cells we used semiquantitative RT-PCR, western blots and immunohistochemistry. The expression of peroxiredoxins I and II was analysed after treatment with cytokines, hydrogen peroxide, alloxan or streptozotocin in the rat insulinoma cells INS-1 using RT-PCR and western blots. Results. We show that peroxiredoxins I and II are present in the cytoplasm of pancreatic islet cells as well as in insulinoma cell lines βTC6-F7 and INS-1. Peroxiredoxins I and II were up-regulated by all stress agents used. Conclusion/interpretation. Beta cells, undersupplied with well characterized antioxidant enzymes, possess an additional antioxidant system which is inducible by oxidative as well as nitrosative stress. [Diabetologia (2002) 45:867-876]

NCI, the C-terminal non-collagenous globular domain of collagen IV, represents one of the two end regions responsible for the assembly and cross-linking of the extracellular network of basement membrane collagen. Several cDNA clones for the NCI domain of the ctl(IV) collagen chain of mouse have been isolated by using synthetic oligonucleotides as screening probes for mouse libraries. The oligonucleotides were synthesized according to known stretches of the corresponding protein sequence. Sequencing of the overlapping cDNA clones allowed the complete amino acid sequence of the NCl domain to be deduced as well as the C-terminal 165 amino acid residues of the triple helix. It consists of 229 amino acid residues which comprise two homologous regions with a high content of cysteine. These DNA and protein sequences are compared to the corresponding sequences of other collagens and discussed with respect to their structural and biological significance.The main collagenous component of basement membranes is collagen IV. The triple helical molecule is 400 nm long and carries a globular domain at its carboxy-terminal [l -31. It contains two ctl (IV) chains and one a2(IV) chain, each consisting of approximately 1700 amino acid residues [4, 51. The triple-helical part of these chains is frequently interrupted by non-helical regions [6, 71, in contrast to interstitial collagen chains. In basement membranes, collagen IV forms a macromolecular network in which the 30-rim-long N-terminal region (7s domain) of four molecules are linked together. Each of these molecules becomes connected at the opposite end, via the C-terminal globule (NCI domain), to the NCI domain of yet another molecule. The network is stabilized by the formation of intermolecular cross-links at both ends [8, 91. This organization differs from the assembly of the interstitial collagens I, I1 and 111, whose 300-nm-long molecules are aligned in parallel but in a D-staggered array (D = 67 nm)While the amino acid sequence of large parts of the triplehelical region of the al(1V) chain from human [7] and mouse [6] is known, the C-terminal globular domain proved to be difficult to characterize, probably because of its hydrophobic character. Since the NCI domain is important for the assembly of the molecules, we decided to determine its amino acid sequence via the corresponding cDNA. Appropriate for such investigations is poly(A)+ RNA from tissues and cell lines which are known to produce basement membranes and type IV collagen, in particular the EHS mouse tumor [l I], the PYS-2 cell line [I, 121 and 1101.both sources contain very little translatable mRNA for the chains of type IV collagen, the amount being estimated to be less than 0.1 % (M. Laurent, I. Oberbaumer, unpublished). However, induced F9 cells have been reported to contain higher levels of these mRNAs [I 51.Here we will present our data on three cDNA clones, two from an EHS library and one from an F9 library, which cover the C-terminal area of the triple helix, the globular domain of the a1 (IV) chai...