One of the primary aims was to understand the relationship between tissue oxygenation and renal parenchyma depth. Our current study revealed two categories of renal oxygenation manifestation patterns. One pattern, the sharp uptrend style, has better tissue oxygenation with a slight fluctuation in the superficial layers of the kidney. Subsequently, a sharp increment of deoxygenation was observed in the deep layers of the renal parenchyma. Although this pattern has similar characteristics with the well-known renal tissue oxygenation feature, such as higher deoxygenation in the medulla than in the cortex [19], a distinct two-phase oxygenation feature was different from that in many previous studies [20-22]. This sharp uptrend style of renal R2* values might correlate with a sophisticated relationship between tissue partial pressure of oxygen (PaO2) and oxyhemoglobin saturation dissociation curve. Tissue oxyhemoglobin saturation could be maintained in a stabilized level when PaO2 is above 60 mmHg, whereas there is a steep gradient with a sharp decrease in oxyhemoglobin when the PaO2 is less than 26.6 mmHg. Previous research has shown that PaO2 in the majority of cortexes is usually higher than 60 mmHg, whereas medullary PaO2 rarely exceeds 26.6 mmHg [23]. Another relevant reason was interrelated with 2,3-diphosphoglycerate (DPG)-mediated oxyhemoglobin affinity. A lessening in oxyhemoglobin affinity has been deemed as an important physiological adaptive response to conditions in which oxygen delivery is impaired. The increased oxyhemoglobin affinity may actually impair tissue oxygenation [24]. Moreover, another pattern of R2* values, the flat uptrend style, was also found in our current study. We found that the R2* values fluctuated in a narrow range throughout the depth of renal parenchyma. R2* values in the deep medulla were only slightly higher than those in the superficial cortex. We even found no discrepancy in R2* level between the superficial renal zone and the deep renal zone, in sporadic samples. This phenomenon implies that tissue oxygenation in the deep medullary zone is not always lower than that in the superficial cortical zone. This discovery is a challenge to the recognized opinion that had been testified by many studies.
Unfortunately, we could not detect renal parenchyma histological and cellular physiological conditions simultaneously. The potential explanation of these two patterns was only based on reasonable ratiocination. In renal cortical zone, the oxygen tension is more variable because of the fluctuation of renal blood perfusion. The existence of an arterial-to-venous oxygen shunt mechanism can maintain the average partial pressure of oxygen (pO2) at approximately 30 mmHg. Schurek et al and Welch et al verified that oxygen tension in renal vein exceeded that in glomerular capillaries and that in the efferent arteriole, respectively [25, 26]. These findings confirmed the existence of oxygen shunt, which was the renal oxygenation homeostatic mechanism. The arterial-to-venous oxygen shunt mechanism maybe one of reasons why deoxyhemoglobin can be detected in the well blood perfusion cortical zone. However, the oxygen supplement in medullary zone was obvious lower than that in cortical zone, not to mention high local oxygen consumption for tubular reabsorption. Other compensative mechanisms such as prostaglandins, nitric oxide (NO), and adenosine were involved. These compensative mechanisms continuously adjust medullary tubular transport activity to the limited available oxygen supply, acting by both enhancement of regional blood flow and downregulation of distal tubular transport, particularly in medullary thick ascending limbs (mTAL) [23]. We speculated the gradient of deoxyhemoglobin in medulla perhaps preserved at similar level in cortex. On the other hand, another pattern of R2* value called “sharp uptrend style” could be also found. It was implied that the quantity of deoxyhemoglobin increased significantly. This R2* values manifestation was observed not only in patients with lupus nephritis but also in healthy volunteers. We speculated several possible pathophysiological mechanisms were involved. Firstly, glomerular hyperfiltration is one condition that increases oxygen demand in renal parenchyma. When glomerular filtration rate is exorbitance, more plasma is also filter out. This condition results in sodium overload in tubular lumen and requires more energy for the reabsorption of sodium by tubular cells[27]. Secondly, oxidative stress can result tissue hypoxia by superoxide radicals decreasing the bioavailability of nitric oxide. Reduced tissue nitric oxide leads to vasoconstriction and decrease in regional blood flow. Furthermore, increased oxidative stress can evaluate oxygen consumption in kidney, possibly via the effects on tubular transporters [28, 29]. Thirdly, loss of peritubular capillaries or reduction in peritubular capillary flow may give raise to renal hypoxia. Previous studies on animal models have showed that tubulointerstitial injury is associated with the distortion and loss of peritubular capillaries[30]. Activation of renin-angiotensin system (RAS) and imbalance of vasoactive substances lead to vasoconstriction and lessen peritubular capillary flow[27].
Our current findings were different from both earlier BOLD MRI studies and direct measurement of tissue oxygen levels with oxygen electrodes animal studies. Our current study found that not all samples from deep medulla showed significant higher R2* values. Compared with R2* values in cortical zone, similar R2* values were detected by BOLD-MRI in deep medullary zone. Moreover, this quaint phenomenon displayed in both patients with LN and healthy controls. It seemed that deep medullary zone had also well oxygenation. This specious deduction contradicted with results for renal oxygenation detected by microelectrode in many previous animal investigations [31, 32]. It seemed dogmatic that higher tissue oxygenation was also found in deep medullary zone according to our observation. In evaluation renal oxygenation status, four kinds of methods were usually applied. (1) measurement of oxidative ability of oxygen molecules, usually by microelectrode. (2) detection the ratio of oxyhemoglobin/total hemoglobin. (3) detection of HIF activity. (4) measurement the magnitude of oxygen molecules in renal parenchyma. Previous animal studies proved difference of oxygen partial pressure between renal cortex and renal medulla. Results in these investigations were obtained by microelectrode in general. BOLD-MRI technique applied R2* values as oxygenation sensor. R2* values which is called spin-spin relaxation rate reflects the deoxyhemoglobin concentration in local tissues. The amount of R2* values dependent on local deoxyhemoglobin concentration rather than local oxygen partial pressure. For example, when renal tissues suffered from anemia, the amount of hemoglobin or deoxyhemoglobin may frequently change. Whereas, the partial pressure of oxygen usually keeps stable state. So, our study team speculated that there was a possibility which ostensible results could be found by BOLD-MRI and microelectrode simultaneously. On the other hand, results of deoxyhemoglobin concentration-based method were influenced by multiple factors such as local blood flow supplementation, changes in pH, acid-base disorder, micro-environmental changes (Bohr effect), oxygen shunt between renal arterial and venous, etc [5, 33]. Our research team speculated one or several mechanisms gave rise to lower deoxyhemoglobin in deep medulla. This may partially interpret why corticomedullary gradient of oxygenation is not observed by R2* index. In order to testify our speculation, another MRI-based technique called relaxation rate of the T1 signal (R1) can be applied, which may correlate with oxygen molecules itself, although it is less sensitive in detecting renal hypoxia than R2*-based technique [34].
Another result of our study involved exploring the possibility of describing renal parenchyma R2* patterns by mathematic models. Some investigators thought that the renal tissue oxygenation status was influenced by multiple factors, such as local blood supplementation and tissue oxygen consumption. Therefore, detected R2* values in each pixel from anywhere in the BOLD images were always the integrated results of multiple physiological factors. The key problem was that the precise participation proportion of each factor was not well understood. However, we believed that information on all involved physiological factors with similar to the encrypted message was still sealed in these R2* maps. We still hope to unlock this sealed information with the right decoding method. By testing the multiple mathematic functions, we found that Gaussian function had the best capability to fit practical R2* data. Moreover, both the sharp uptrend style data and the flat uptrend style data fit the Gaussian function well with two or more compartments. Although there was no reliable evidence to verify the corresponding relationship between renal biological factors and compartments of fit Gaussian functions, we hypothesized that those multiple encrypted and perplexing physiological messages in the R2* data could be transformed into another mode. Perhaps we will decode the encrypted biological messages that were composed in R2* data by studying fit Gaussian functions.
Our study also compared renal histological data with R2* values. In proliferative LN such as type III or type IV, lower renal tissue R2* values usually correspond with severe tubular damage. The inverse manifestation was observed in non-proliferative LN in which mild tubular injuries could be confirmed with renal biopsy samples [35]. Despite statistical discrepancy of R2* values detected between the two groups in both sharp uptrend and flat uptrend style data, the substantial R2* difference was very small. We speculated that at least two factors were involved and that the pathogenesis was similar to that in patients with renal artery stenosis. Hansell et al. found that kidneys usually had increased renal blood flow instead of regional pO2 when the renal parenchyma was under severe hypoxia conditions. The increased renal blood flow also led to GFR increments that subsequently induced more filtered sodium in the renal tubules. The reinforced tubular sodium reabsorption was the main reason for increased oxygen consumption [36]. Due to R2* values indicated magnitude of deoxyhemoglobin according to the principle of BOLD imaging, lower R2* in cortex implied the better cortical oxygenation. Morphology assessment of renal biopsy samples showed tubulointerstitial inflammation or atrophy could be been in patients with proliferative lupus nephritis. We speculated these pathological injuries might weaken tubular transport activity, which subsequently reduced oxygen consumption and decreased the R2* values. Our surmise was also confirmed by our previous study. Furthermore, faded GFR also led to downward R2* due to lessened solute delivery for tubular transport. These possible mechanisms listed above might explain the tendency of R2* in Figure 3 that both crossover of two R2* curves in medulla and separation of two R2* curves in cortex could be detected simultaneously. Our research team also considered that the integrity of renal physiological function in healthy volunteers was superior to that in patients with lupus nephritis. The preserved glomerular and tubulointerstitial construction and function were prone to oxygen consumption, and integrate peritubular capillary would transport more hemoglobin into renal tissues. We speculated that this was one of the reasons why higher R2* values were found at deep medullary zone in sharp upturn style and superficial cortical zone in flat upturn style.
We also developed a novel ROI method called the “narrow rectangle” or “virtual probe” to measure renal R2* values. This was the principal reason why we devised and applied this special ROI technique. Renal microstructure displays distinct directional characteristics and spatial heterogeneity. Renal parenchyma oxygenation features are based on its histological anatomy foundation. The well-known opinion that the degree of renal blood oxygen saturation in the cortical zone is always higher than that in the medullary zone is based on previous ROI analysis methods such as regional ROI selection [37], compartmental approach [38], fractional kidney hypoxia [39], CO [7], and TLCO [8]. However, investigators either handled nonconsecutive crude R2* data by regional ROI selection or acquired calculated mean R2* results by CO and TLCO methods. These inherent shortcomings remained an obstacle to the procurement of the explicit oxygenation tendency throughout the renal parenchyma. Contrary to previous conventional ROI analysis methods, our new analysis technique revealed that deep medullary oxygenation was not always overtly lower than cortical oxygenation. This unusual discovery challenged the well-known opinion.
Our current study still had several shortcomings. First, our study merely investigated renal R2* signal instead of other functional MR signals such as ADC, synchronously. We only knew the existing oxygenation condition, whereas we did not understand the precise mechanism of hypoxia status formation. Second, the meaning of fitted Gaussian functions had not yet been explained or proved by other physiological studies, and we only hypothesized that fitted Gaussian formulas represent renal hypoxia outline in the kidney cortical and medullary zones. Third, the narrow rectangular ROI analysis method that was adopted in our study had not been tested by many researchers. Because the highly observer-dependent property of previous conventional ROI analysis methods has been proved, we did not know the new ROI analysis technique had the highly reproducible and lower variant characteristic.