Binding of pheromones to urinary proteins of rats and/or mice is increasingly becoming recognised as having complex biological roles in social communication and heterosexual interaction [1–3].

In the 1980s, search for organic substitutes for the lead tetra-ethyl added to the petrol of motor vehicles recognised several hydrocarbons of interest. However, toxicity tests revealed renal carcinogenic properties in male rats and further study showed a correlation with histopathological changes in kidney cortex. Hyaline droplets within nephron epithelia were attributed to accumulation of the putative carcinogen bound to a protein (α2u-globulin) the size of which allowed excretion in glomerular filtrate. Normal digestion of the protein in proximal tubule epithelia became inhibited when an isoprenoid hydrocarbon (e.g. d-limonene) is bound to the protein. The consequent intracellular accumulation of hyaline droplets caused epithelial cell necrosis and cell proliferation in response to injury. This non-genotoxic mechanism for rat renal carcinogenesis was a convenient finding, since attitude in human cancer risk assessment is less severe if a chemical carcinogen is shown to act via a mechanism that does not involve covalent binding to DNA [4].

α2u-globulin had long been known as a major urinary protein in male rats [5] and its role in rat experimental nephropathy was a timely finding in hydrocarbon toxicology. It was a topic of much toxicological discussion in the 1990s [6–9]. However, recent comprehensive review of the pathological connection between α2u-globulin nephropathy and renal tumours across many studied examples could not be so sure of a simple causal relationship [10].

The seminal studies of Roy et al. [11] resolved five isoelectric forms of α2u-globulin by 2-D electrophoresis and showed that the most prominent isoform with a pI of 6.1 was the first to appear in Fischer males at puberty (40 days old). Radioimmunoassay showed that, relative to total protein in liver, α2u-globulin ultimately rose to a maximum ~ 3.5 ng/mg at 70 days old. Subsequently, α2u-globulin was isolated from kidney cytosol of adult Fischer male rats and remained a single entity in SDS-PAGE and capillary electrophoresis [12], but occurred in greater concentration in kidney of rats given agents that induce α2u-globulin-nephropathy. However, α2u-globulin had already been described as resolvable electrophoretically into two distinct molecular forms [13].

All rats also produce another form of constitutive α2u-globulin, synthesised in the submaxillary gland via a set of genes that are different from those for the form in liver [14], but the salivary form is unlikely to complicate the picture in urine. The lipocalin is also found in lachrymal glands [15].

A possible extension of the range of hydrocarbons that can form biologically-active ligands with α2u-globulin to include ochratoxin A has been studied to determine whether protein droplet nephropathy is a prelude to the renal tumours that ochratoxin A can cause in male rats [16]. Clearly this nephropathy is not involved. However, binding of ochratoxin A to urinary α2u-globulin has since been seen [17] on PhastGel electrophoretograms (although with a 8-25% gradient), which could indicate potential for assisted nephron uptake of ochratoxin A during α2u-globulin salvage in proximal segments. Understanding the onset of such in young rats in toxicology research would be a factor in optimal experimental design. The crystal structure of α2u-globulin has been determined [18] and peptide sequences for others are available, but no study of occurrence during early puberty had yet been made.

For economic reasons experimental toxicology research often uses quite young rodents, in which toxicokinetics of xenobiotics may be influenced by small proteins circulating in blood. Currently there is only sparse data on this topic. The present pilot study has thus explored temporal changes in the small urinary protein pattern in two strains of laboratory rat during early puberty, collected without any stressful intervention, together with measurement of urinary testosterone as an indicator of expanding maleness. Several lipocalins have been partially characterised according to some of their peptide sequences after tryptic digestion.

Excision of each band in the present gels sought to make a balance between both comprehensive recognition and demonstration of the principal component(s). Close electrophoretic mobilities of all these proteins means that examples could have appeared in one band in one time-sample and in an adjacent one in another.

Nevertheless, resolution of several rat urinary proteins in the 18 kDa range, some of which appear identical, in PhastSystem mini-gels still leaves open the possibility of a range of post-translational forms [20] which could include glycosylation. Use of a high protein diet in young rats did not appear to result in any marked change in urinary protein pattern but was associated with slightly earlier appearance of a 4-band lipocalin pattern. However, more abundant dietary protein might have diminished lipocalin salvage efficiency within nephrons, reflected in a greater abundance of the proteins in urine.

Considering the classical literature on α2u-globulin, comparison of its tissue-specific RNA from liver and both salivary and lachrymal glands in Sprague-Dawley rats Gubits et al. [15] found the RNA for the latter pair already in both sexes at 6 days old, whereas only at 44 days in liver. Kulkarni et al., from the same laboratory [21], also reported that 44 days was the stage at which a significant α2u-globulin transcription rate became evident. Also, administration (i.p.) of 17β-estradiol daily to male rats virtually eliminated hepatic α2u-globulin transcription within 8 days. Dot blot analysis of liver RNA from the same rat strain via hybridisation to nick-translated α2u-globulin cDNA gave no signal at 34 days of age, but a positive signal at and after 44 days. Findings in the present study (Figure 3) are consistent with this, but revealed other rat lipocalins in urine from 36 days old. Clearly there is much to understand about regulation of lipocalin synthesis in juveniles.

Roy et al. [11] recognised an iso-electric form of α2u-globulin by radioimmunoassay as early as 40 days old in the liver of Fischer rats. It is tempting to equate this with the ‘rat urinary protein’ observed first here in Sprague-Dawley rats, but Roy et al. [11] regarded this as the principal α2u-globulin. However, we presently resolve this into two forms. These may equate to forms A and B, previously recognised [13]. According to Roy et al. [5], maximum incidence of this compound occurred at 70 days, declining in later life. Otherwise it is difficult to equate any of the present urinary compounds defined by their amino acid sequence data with previous literature findings. This is partly due to the good resolution achieved in the PhastGels, but of course urinary content is only what remains after some of the glomerular-filterable lipocalins in blood plasma have been absorbed by proximal nephron epithelia for salvage of the nitrogen component. Nevertheless, non-invasive sampling gave insight into qualitative changes in circulating composition of lipocalins during early puberty and also demonstrated subsequent increased abundance of the two forms of these proteins in the ~18 kDa range. Relative molecular masses, assessed electrophoretically, were quoted as 18,800 and 18,100 [13], which could be accommodated in parts of the peptide sequence in Figure 8 that are unrecognised in the present tryptic digest protocol. Alternatively, post-translational modifications alone may accounts for the slightly different electrophoretic mobilities.

Biological significance of rat urinary proteins 1 and 2 is not yet known. However, recognition of rat urinary protein 1 in our reference sample of adult rat α2u-globulin, isolated originally at UK’s Imperial Chemical Industries (interested in light hydrocarbon nephropathy but before any biological function was known) in the 1980s [22], links the present study into a historical toxicology context and implies the former’s persistence as a minor urinary protein at least into adult (12-14 week old) life. Notably in [22], three isoforms of a generic α2u-globulin were recognised in 2D-PAGE of liver extracts of 32 outbred Wistar-derived rats. One isoform was common to all, but three combinations were recognised. One of these pairs characterised liver of 8 inbred F344 rats. However, pooled urine from Wistars contained all three α2u-globulin isoforms. From the findings of Elliott et al. [22] assumption of similarity of urinary lipocalin pattern of F344 rats in the present study with the other outbred strains requires structural conformation. Much remains to be understood.

Similar testosterone concentration, expressed relative to excretion of creatinine, on days 49 and 57 in Wistar males, and over the 36-56 day period in Sprague-Dawley animals on a high protein diet, demonstrated circulation of androgen already in the youngest animals. Over these periods there appeared to be a marked change in urinary protein composition, switching from rat urinary proteins alone to including Major rat urinary protein (Figures 3 and 5). This suggests that the latter’s appearance was not driven directly by a concentration surge of circulating androgen. However, apparent increase in overall abundance of urinary proteins by day 107 was clearly concomitant with the several-fold increase in circulating testosterone, if urine evidence at least roughly reflects the situation in its blood plasma source. Nevertheless, the present urine values of ~ 6 ng/ml are close to the 4 ng/ml reported for whole blood by radioimmunoassay in 6 month old Dark Agouti, Wistar and Sprague-Dawley males [23], and ~ 5 ng/ml, also by radioimmunoassay, in adult Sprague-Dawley rat serum [24]. However, we recognise that any further monitoring of circulating testosterone, particularly in pre-pubertal rats, should optimise urine sample collection to avoid any evaporation.

Although androgen-dependence as a classical descriptor for rat α2u-globulin is not questioned, the context and mechanism for this is unclear. In a recent publication [25] serum α2u-globulin concentration was measured (~ 50 μg/ml) 24 days after surgical castration, but unfortunately a value for entire rats at the same stage was not given. However, it was implied that although marked endocrine disruption for this lipocalin had occurred, it had not been complete. We deduce that androgen synthesis in adrenal glands may have sustained some hepatic α2u-globulin synthesis. Notably in the same context, daily sub-cutaneous testosterone replacement (0.5 mg/kg b.w.) was necessary significantly to increase α2u-globulin circulation 4-fold for similar castrates. Although sub-cutaneous, this seems a rather high testosterone dosage to equate approximately to the present 6 ng/ml urinary testosterone in Wistar males (Table 1, estimated ~ 60 ng excreted daily) that was adequate for maintaining abundant urinary α2u-globulin. Again, there is much to understand about androgen influence in rat hepatic α2u-globulin synthesis and in urinary lipocalins in general.

It is reasonable to perceive that, since steady-state plasma concentration of the xenobiotic ochratoxin A in female rats seems to be significantly higher than in males given equivalent chronic dietary doses [26], experimentally-impaired hormonal maleness in males could reduce this gender difference. Therefore, we cannot ignore findings of ongoing studies concerning gender-related differential toxicological responses of rats to chronic dietary exposure to ochratoxin A in which experimental interventions that markedly diminish circulating testosterone closed the gap between male and female titres for ochratoxin A in plasma as male values increased. Hepatic expression of α2u-globulin synthesis in rats is strictly androgen-dependent but is strongly suppressed by oestrogen [27]. Concurrent with the present study, the immunoblotting protocol of Mantle and Nagy [17] was revisited by us, although necessarily using a different ochratoxin A antibody, also sourced from R-Biopharm Rhone Ltd., Glasgow, UK. However, binding of ochratoxin A to α2u–globulin could not be confirmed. Both antibodies had been produced commercially specifically for the production of immuno-affinity columns, initially sourced from animals but latterly replaced by one from fermentation. Although when immobilised on the immunoaffinity column’s support substrate both were very efficient for selectively binding ochratoxin A, there could have been no expectation that the second antibody as a free protein would have had a binding domain for α2u–globulin, as had been apparent concerning the first-used antibody. Therefore, at present it has neither been possible to confirm nor to disprove the findings and perceptions of Mantle and Nagy [17]. However, favourable competition in circulating blood between a small lipocalin and serum albumin in binding ochratoxin A in males could provide a mechanism for maintaining a lower circulating OTA concentration than in comparable females. If a component of the α2u-globulin complex binds ochratoxin A this would be in dynamic competition with the well-known binding to serum albumin, and the ligand would easily take some toxin out of circulation with each pass through glomeruli in males and excrete via nephrons. Of course there would initially have been an interesting brief competition between small and large proteins in the hepatic portal vein for newly-absorbed ochratoxin A molecules before meeting newly-elaborated α2u-globulins in liver. Thus, a better understanding of the identity and kinetics of urinary lipocalins from early puberty seems to be required. Putative binding of ochratoxin A to a rat lipocalin now requires verification, hopefully via selection of a suitable ochratoxin A antibody from the many products available commercially. The structural conformation of this antibody must be able to retain its binding capacity to ochratoxin A even when part of the latter is located in a pocket domain of a lipocalin such as an α2u-globulin. Unfortunately the original antibody used by Mantle and Nagy [17] no longer exists.

We also note that, concerning the toxicological example of ochratoxin A discussed above, the immunoblot findings [17] were observed in urine of male F344 and F344 x Sprague-Dawley F₁ hybrid rats. Earlier, F344 rats differed qualitatively in ‘α2u-globulin isoform’ composition from that of Sprague-Dawley or Wistar-derived strains [22]. F344 rats are favoured for some toxicology studies because they tend to reveal a worst-case scenario and were used in the 1989 US National Toxicology Programme study [28] revealing striking renal tumourigenesis in males in response to lifetime exposure to ochratoxin A, and in other studies cited in [17]. Wistar and Sprague-Dawley rats have not been used for lifetime renal tumourigenesis experiments with ochratoxin A. Therefore a question arises as to whether the immunoblot findings [17] were influenced by an F344 genome specificity for circulating lipocalins and might make F344 responses atypical for all rats. Unquestioning extrapolation from F344 responses to humans could then be unsafe.

Convenience collection of small urine samples proved to be an ethical, economic, effective and non-intrusive source for monitoring dynamics both of small proteins and testosterone in growing rats. For the proteins, PhastGel separation seems to be method of choice. The pilot study allowed us to conclude that the qualitative change in urinary protein composition is probably reflective of similar temporal dynamics in hepatic synthesis and in blood. However, dynamics in blood would be much more difficult to observe in plasma because of its greater complexity. Consequently, resolution of major rat protein (α2u-globulin) into two components raises a question whether potential for binding of xenobiotics applies to both forms.

Whereas PhastGel separation of small urinary proteins already revealed complexity, further development of gradient separation should allow refined trypsin digest data on the homogeneity of bands. The present gels were a technical challenge in excision of narrow bands, and we chose examples with minimal amounts of proteins. Other significant peptide fragment patterns, attributable to as yet unrecognised proteins, could be revealed from study of less well resolved bands containing more protein. We conclude that proteomics may reveal a richer array of rodent urinary proteins than previously thought.

Expanded understanding of the dynamics of lipocalin synthesis, circulation, excretion and occurrence in urine, not only as young males enter puberty but also through adult life, has potential in explaining marked gender difference in response to some xenobiotic toxins where the mechanism yet remains obscure. We conclude that such potential should be explored further.